okadaic-acid and fostriecin

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

Adenosine signaling via A1 receptor (A1R) and A2A receptor (A2AR) has shown promise in revealing potential targets for neuroprotection in cerebral ischemia. We recently showed a novel mechanism by which A1R activation with N(6)-cyclopentyl adenosine (CPA) induced GluA1 and GluA2 AMPA receptor (AMPAR) endocytosis and adenosine-induced persistent synaptic depression (APSD) in rat hippocampus. This study further investigates the mechanism of A1R-mediated AMPAR internalization and hippocampal slice neuronal damage through activation of protein phosphatase 1 (PP1), 2A (PP2A), and 2B (PP2B) using electrophysiological, biochemical and imaging techniques. Following prolonged A1R activation, GluA2 internalization was selectively blocked by PP2A inhibitors (okadaic acid and fostriecin), whereas inhibitors of PP2A, PP1 (tautomycetin), and PP2B (FK506) all prevented GluA1 internalization. Additionally, GluA1 phosphorylation at Ser831 and Ser845 was reduced after prolonged A1R activation in hippocampal slices. PP2A inhibitors nullified A1R-mediated downregulation of pSer845-GluA1, while PP1 and PP2B inhibitors prevented pSer831-GluA1 downregulation. Each protein phosphatase inhibitor also blunted CPA-induced synaptic depression and APSD. We then tested whether A1R-mediated changes in AMPAR trafficking and APSD contribute to hypoxia-induced neuronal injury. Hypoxia (20 min) induced A1R-mediated internalization of both AMPAR subunits, and subsequent normoxic reperfusion (45 min) increased GluA1 but persistently reduced GluA2 surface expression. Neuronal damage after hypoxia-reperfusion injury was significantly blunted by pre-incubation with the above protein phosphatase inhibitors. Together, these data suggest that A1R-mediated protein phosphatase activation causes persistent synaptic depression by downregulating GluA2-containing AMPARs; this previously undefined role of A1R stimulation in hippocampal neuronal damage represents a novel therapeutic target in cerebral ischemic damage.

Topics: Animals; Excitatory Postsynaptic Potentials; Furans; Hippocampus; Lipids; Male; Neurons; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Protein Transport; Pyrones; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, AMPA; Reperfusion Injury; Synaptic Transmission; Tacrolimus

Ciliary muscle is a smooth muscle characterized by a rapid response to muscarinic receptor stimulation and sustained contraction. Although it is evident that these contractions are Ca(2+)-dependent, detailed molecular mechanisms are still unknown. In order to elucidate the role of Ser/Thr protein phosphatase 2A (PP2A) in ciliary muscle contraction, we examined the effects of okadaic acid and other PP2A inhibitors on contractions induced by carbachol (CCh) and ionomycin in bovine ciliary muscle strips (BCM). Okadaic acid inhibited ionomycin-induced contraction, while it did not cause significant changes in CCh-induced contraction. Fostriecin showed similar inhibitory effects on the contraction of BCM. On the other hand, rubratoxin A inhibited both ionomycin- and CCh-induced contractions. These results indicated that PP2A was involved at least in ionomycin-induced Ca(2+)-dependent contraction, and that BCM had a unique regulatory mechanism in CCh-induced contraction.

Topics: Animals; Calcium; Carbachol; Cattle; Ciliary Body; Enzyme Inhibitors; In Vitro Techniques; Ionomycin; Muscle Contraction; Muscle, Smooth; Mycotoxins; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Pyrones

We recently reported that microinjection of ethanol into the rostral ventrolateral medulla (RVLM) elicits modest increases in local extracellular signal-regulated kinase (ERK) and blood pressure (BP) in conscious normotensive rats. In this study, we tested the hypothesis that RVLM ser/thr phosphatases dampen the ERK-dependent pressor effect of ethanol in normotensive rats. We show that the pressor response elicited by intra-RVLM ethanol (10 μg) was (i) abolished following local ERK inhibition with PD98059 (1 μg) and (ii) associated with significant reduction in local phosphatase activity. Inhibition of the RVLM ser/thr phosphatase activity by okadaic acid (OKA, 0.4 μg) or fostriecin (15 pg) caused significant increases in blood pressure (BP) and potentiated the magnitude and duration of the pressor response as well as the phosphatase inhibition elicited by subsequent intra-RVLM administration of ethanol. Intra-RVLM acetaldehyde (2 μg), the main metabolic product of ethanol, caused no changes in BP or RVLM phosphatase activity but it produced significant increases in BP and inhibition of local phosphatase activity in rats treated with OKA or fostriecin. Together, the RVLM phosphatase activity acts tonically to attenuate the ERK-dependent pressor effect of ethanol or acetaldehyde in normotensive rats.

Topics: Acetaldehyde; Animals; Blood Pressure; Enzyme Inhibitors; Ethanol; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Male; Medulla Oblongata; Okadaic Acid; Phosphoric Monoester Hydrolases; Polyenes; Pyrones; Rats, Inbred WKY; Vasoconstrictor Agents

Our recent studies on human airway serous-like Calu-3 cells showed that cAMP agonists stimulated a HCO3(-) rich secretion containing up to 80 mM HCO3(-). This alkaline secretion relied on a coordinated switch in the activity of distinct Cl(-)-HCO3(-) anion exchangers (AE) located at different regions of the cell. At the apical membrane, cAMP agonists activated the electroneutral AE pendrin (SLC26A4), together with cystic fibrosis transmembrane conductance regulator (CFTR), while at the basolateral membrane the agonists inhibited AE2 (SLC4A2). However, the underlying mechanism(s) that orchestrates this cAMP-dependent switch in AE activity has not been elucidated.. Apical and basolateral Cl(-)-HCO3(-) exchange was assessed by measuring Cl(-)-dependent changes in intracellular pH (pH(i)).. We show that protein phosphatase 1 (PP1), together with CFTR, play central roles in this reciprocal regulation of AE activity. Activation of pendrin by cAMP agonists, but not inhibition of the basolateral exchanger, was protein kinase A-dependent. Knocking down CFTR expression, or blocking its activity with GlyH-101, led to incomplete inhibition of the basolateral AE by cAMP, supporting a role for CFTR in this process. Addition of the PP1/2A inhibitor, okadaic acid, but not the PP2A specific inhibitor fostreicin, mimicked the effect of cAMP stimulation. Furthermore, okadaic acid-treated Calu-3 monolayers produced a more alkaline fluid than untreated cells, which was comparable with that produced by cAMP stimulation.. These results identify PP1 as a novel regulator of AE activity which, in concert with CFTR, coordinates events at both apical and basolateral membranes, crucial for efficient HCO3(-) secretion from Calu-3 cells.

Topics: Bicarbonates; Cell Line, Tumor; Cell Membrane; Chloride-Bicarbonate Antiporters; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Inhibitors; Glycine; Humans; Hydrazines; Membrane Transport Proteins; Okadaic Acid; Polyenes; Protein Phosphatase 1; Pyrones; Respiratory System; Sulfate Transporters

CaBP4 is a neuronal Ca(2+)-binding protein that is expressed in the retina and in the cochlea, and is essential for normal photoreceptor synaptic function. CaBP4 is phosphorylated by protein kinase C zeta (PKCζ) in the retina at serine 37, which affects its interaction with and modulation of voltage-gated Ca(v)1 Ca(2+) channels. In this study, we investigated the potential role and functional significance of protein phosphatase 2A (PP2A) in CaBP4 dephosphorylation.. The effect of protein phosphatase inhibitors, light, and overexpression of PP2A subunits on CaBP4 dephosphorylation was measured in in vitro assays. Pull-down experiments using retinal or transfected HEK293 cell lysates were used to investigate the association between CaBP4 and PP2A subunits. Electrophysiologic recordings of cotransfected HEK293 cells were performed to analyze the effect of CaBP4 dephosphorylation in modulating Ca(v)1.3 currents.. PP2A inhibitors, okadaic acid (OA), and fostriecin, but not PP1 selective inhibitors, NIPP-1, and inhibitor 2, block CaBP4 dephosphorylation in retinal lysates. Increased phosphatase activity in light-dependent conditions reverses phosphorylation of CaBP4 by PKCζ. In HEK293 cells, overexpression of PP2A enhances the rate of dephosphorylation of CaBP4. In addition, inhibition of protein phosphatase activity by OA increases CaBP4 phosphorylation and potentiates the modulatory effect of CaBP4 on Ca(v)1.3 Ca(2+) channels in HEK293T cells.. This study provides evidence that CaBP4 is dephosphorylated by PP2A in the retina. Our findings reveal a novel role for protein phosphatases in regulating CaBP4 function in the retina, which may fine tune presynaptic Ca(2+) signals at the photoreceptor synapse.

Topics: Adaptation, Ocular; Animals; Calcium Channels, L-Type; Calcium-Binding Proteins; Dark Adaptation; Enzyme Inhibitors; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Models, Biological; Nerve Tissue Proteins; Okadaic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Protein Kinase C; Protein Phosphatase 2; Protein Phosphatase 2C; Pyrones; Retina; Signal Transduction

The G(i)-coupled somatostatin 2A receptor (sst2A) mediates many of the neuromodulatory and neuroendocrine actions of somatostatin (SS) and is targeted by the SS analogs used to treat neuroendocrine tumors. As for other G protein-coupled receptors, agonists stimulate sst2A receptor phosphorylation on multiple residues, and phosphorylation at different sites has distinct effects on receptor internalization and uncoupling. To elucidate the spatial and temporal regulation of sst2A receptor phosphorylation, we examined agonist-stimulated phosphorylation of multiple receptor GPCR kinase sites using phospho-site-specific antibodies. SS increased receptor phosphorylation sequentially, first on Ser-341/343 and then on Thr-353/354, followed by receptor internalization. Reversal of receptor phosphorylation was determined by the duration of prior agonist exposure. In acutely stimulated cells, in which most receptors remained on the cell surface, dephosphorylation occurred only on Thr-353/354. In contrast, both Ser-341/343 and Thr-353/354 were rapidly dephosphorylated when cells were stimulated long enough to allow receptor internalization before agonist removal. Consistent with these observations, dephosphorylation of Thr-353/354 was not affected by either hypertonic sucrose or dynasore, which prevent receptor internalization, whereas dephosphorylation of Ser-341/343 was completely blocked. An okadaic acid- and fostriecin-sensitive phosphatase catalyzed the dephosphorylation of Thr-353/354 both intracellularly and at the cell surface. In contrast, dephosphorylation of Ser-341/343 was insensitive to these inhibitors. Our results show that the phosphorylation and dephosphorylation of neighboring GPCR kinase sites in the sst2A receptor are subject to differential spatial and temporal regulation. Thus, the pattern of receptor phosphorylation is determined by the duration of agonist stimulation and compartment-specific enzymatic activity.

Topics: Amino Acid Substitution; Animals; CHO Cells; Cricetinae; Cricetulus; Enzyme Inhibitors; G-Protein-Coupled Receptor Kinases; Humans; Mutation, Missense; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Pyrones; Receptors, Somatostatin

Mitotic chromosomal assembly in vertebrates is regulated by condensin I and condensin II, which work cooperatively but have different chromosomal localization profiles and make distinct mechanistic contributions to this process. We show here that protein phosphatase 2A (PP2A), which interacts with condensin II but not condensin I, plays an essential role in targeting condensin II to chromosomes. Unexpectedly, our data indicate that PP2A acts as a recruiter protein rather than a catalytic enzyme to target condensin II to chromosomes. This recruiting activity of PP2A was inhibited by okadaic acid, but not by fostriecin, even though both molecules strongly inhibited the catalytic activity of PP2A. Additionally, we found that the chromokinesin KIF4a is also targeted to chromosomes via the noncatalytic activity of PP2A. Thus, our studies reveal a previously unknown contribution of PP2A to chromosome assembly.

Topics: Adenosine Triphosphatases; Alkenes; Animals; Cell Line; Cells, Cultured; Chromosomes; DNA-Binding Proteins; Enzyme Inhibitors; Humans; Kinesins; Models, Biological; Multiprotein Complexes; Okadaic Acid; Polyenes; Protein Phosphatase 2; Pyrones; Xenopus

The microbial toxin okadaic acid (OA) specifically inhibits PPP-type ser/thr protein phosphatases. OA is an established tumor promoter with numerous cellular effects that include p53-mediated cell cycle arrest. In T51B rat liver epithelial cells, a model useful for tumor promotion studies, p53 activation is induced by tumor-promoting (low nanomolar) concentrations of OA. Two phosphatases sensitive to these concentrations of OA, PP2A and protein phosphatase 5 (PP5), have been implicated as negative regulators of p53. In this study we examined the respective roles of these phosphatases in p53 activation in non-neoplastic T51B cells. Increases in p53 activity were deduced from levels of p21 (cip1) and/or the rat orthologue of mdm2, two p53-regulated gene products whose induction was blocked by siRNA-mediated knockdown of p53. As observed with 10 nM OA, both phospho-ser15-p53 levels and p53 activity were increased by 10 microM fostriecin or SV40 small t-antigen. Both of these treatments selectively inhibit PP2A but not PP5. siRNA-mediated knockdown of PP2A, but not PP5, also increased p53 activity. Finally, adenoviral-mediated over-expression of an OA-resistant form of PP5 did not prevent increased phospho-ser15-p53, p53 protein, or p53 activity caused by 10 nM OA. Together these results indicate that PP5 blockade is not responsible for OA-induced p53 activation and G1 arrest in T51B cells. In contrast, specific blockade of PP2A mimics p53-related responses to OA in T51B cells, suggesting that PP2A is the target for this response to OA.

Topics: Alkenes; Amino Acid Sequence; Animals; Antigens, Viral, Tumor; Cell Proliferation; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; G1 Phase; Liver; Molecular Sequence Data; Nuclear Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Protein Phosphatase 2; Pyrones; Rats; RNA, Small Interfering; Tumor Suppressor Protein p53

Protein kinases and phosphatases catalyze opposing reactions of phosphorylation and dephosphorylation, which may modulate the function of crucial signaling proteins in central nervous system. This is an important mechanism in the regulation of intracellular signal transduction pathways in nociceptive neurons. To explore the role of protein phosphatase in central sensitization of spinal nociceptive neurons following peripheral noxious stimulation, using electrophysiological recording techniques, we investigated the role of two inhibitors of protein phosphatase type 2A (PP2A), fostriecin and okadaic acid (OA), on the responses of dorsal horn neurons to mechanical stimuli in anesthetized rats following intradermal injection of capsaicin. Central sensitization was initiated by injection of capsaicin into the plantar surface of the left paw. A microdialysis fiber was implanted in the spinal cord dorsal horn for perfusion of ACSF and inhibitors of PP2A, fostriecin and okadaic acid. We found that in ACSF pretreated animals, the responses to innocuous and noxious stimuli following capsaicin injection increased over a period of 15 min after injection and had mostly recovered by 60 min later. However, pre- or post-treatment with the phosphatase inhibitors, fostriecin or OA, significantly enhanced the effects of capsaicin injection by prolonging the responses to more than 3 hours. These results confirm that blockade of protein phosphatase activity may potentiate central sensitization of nociceptive transmission in the spinal cord following capsaicin injection and indicate that protein phosphatase type 2A may be involved in determining the duration of capsaicin-induced central sensitization.

Topics: Afferent Pathways; Alkenes; Animals; Capsaicin; Disease Models, Animal; Enzyme Inhibitors; Inflammation Mediators; Male; Nociceptors; Okadaic Acid; Pain; Pain Threshold; Phosphoprotein Phosphatases; Physical Stimulation; Polyenes; Posterior Horn Cells; Pyrones; Rats; Rats, Sprague-Dawley; Reaction Time; Synaptic Transmission; Time Factors

Mitosis and cytokinesis are highly coordinated in eukaryotic cells. But procyclic-form Trypanosoma brucei under G1 or mitotic arrest is still capable of dividing, resulting in anucleate daughter cells (zoids). Okadaic acid (OKA), an inhibitor of protein phosphatases PP1 and PP2A, is known to inhibit kinetoplast replication and cell division yielding multinucleate cells with single kinetoplasts. However, when OKA was applied to cells arrested in G1 or G2/M phase via RNAi knockdown of specific cdc2-related kinases (CRKs), DNA synthesis and nuclear division were resumed without kinetoplast replication or cell division, resulting in multinucleate cells as in the wild type. Cells arrested in G2/M via depleting the mitotic cyclin CycB2 or an aurora B kinase homologue TbAUK1 were, however, not released by OKA treatment. The phenomenon is thus similar to the OKA activation of Cdc2 in Xenopus oocyte by inhibiting PP2A [Maton, et al., Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A. J. Cell Sci. 118 (2005) 2485-2494]. A simultaneous knockdown of the seven PP1s or the PP2A catalytic subunit in T. brucei by RNA interference did not, however, result in multinucleate cells. This could be explained by assuming a negative regulation, either directly or indirectly, of CRK by an OKA-sensitive phosphatase, which could be a PP2A as in the Xenopus oocyte and a positive regulation of kinetoplast replication by an OKA-susceptible protein(s). Test of a PP2A-specific inhibitor, fostriecin, on cells arrested in G2/M via CRK depletion or a knockdown of the PP2A catalytic subunit from the CRK-depleted cells both showed a partial lift of the G2/M block without forming multinucleate cells. These observations support the abovementioned assumption and suggest the presence of a novel OKA-sensitive protein(s) regulating kinetoplast replication that still remains to be identified.

Topics: Alkenes; Animals; CDC2 Protein Kinase; Cell Cycle; Enzyme Inhibitors; Humans; Isoenzymes; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Protein Phosphatase 2; Protein Subunits; Protozoan Proteins; Pyrones; RNA Interference; Trypanosoma brucei brucei

Transcription factor NF-kappaB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF). In the absence of TNF, NF-kappaB is sequestered in the cytoplasm by inhibitory IkappaB proteins. Phosphorylation of IkappaBby the beta-catalytic subunit of IKK, a multicomponent IkappaB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-kappaB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKbeta, which greatly stimulates IkappaB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IkappaB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121-179 of the IKKgamma regulatory subunit. Third, deletion of the PP2A-binding site in IKKgamma attenuates T loop phosphorylation and catalytic activation of IKKbeta in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK.PP2A complexes is required for the proper induction of IkappaB kinase activity in vivo.

Topics: Active Transport, Cell Nucleus; Adenosine Triphosphate; Alkenes; Animals; B-Lymphocytes; Catalysis; Cell Line; Chromatography, Liquid; Cytoplasm; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Gene Deletion; Humans; I-kappa B Kinase; Immunoblotting; Immunoprecipitation; Inflammation; Jurkat Cells; Mice; Mice, Inbred C57BL; Mutation; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Protein Phosphatase 2; Pyrones; Sepharose; Serine; Signal Transduction; Spleen; Threonine; Time Factors; Transfection; Tumor Necrosis Factor-alpha

The viral replication rate in patients infected with human immunodeficiency virus type 1 (HIV-1) is controlled in part by regulation of the transcription of viral genes. The rate of transcription is determined by a complex interplay between cellular and viral proteins and the promoter elements found in the long terminal repeats. Protein phosphatase 2A (PP2A) is a phosphoprotein that plays important roles in the regulation of signal transduction and cell growth. In this report, we demonstrate that overexpression of the catalytic subunit of protein phosphatase 2A (PP2Ac) increases the basal activity of the HIV-1 promoter and, especially, enhances the promoter's response to the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (PMA). Additionally, ectopic PP2Ac enhances activation of HIV-1 provirus by PMA. Okadaic acid, a potent inhibitor of PP2A, markedly reduces both HIV-1 enhancer and proviral activation. Fostriecin, a PP2A inhibitor which has been used as an antineoplastic agent in clinical trials, is also able to inhibit PMA-stimulated HIV-1 proviral activation. These observations demonstrate a role for the important cellular phosphatase PP2A in HIV-1 transcription and replication and also suggest that PKC can potentiate the activity of PP2A. PP2A is a potential target for therapeutic intervention in patients infected with HIV-1.

Topics: Alkenes; HIV-1; Humans; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Promoter Regions, Genetic; Protein Kinase C; Protein Phosphatase 2; Pyrones; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; U937 Cells; Virus Activation

The gap junction protein connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43-46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1-like phosphatase(s).

Topics: Alkenes; Animals; Blotting, Western; Cells, Cultured; Connexin 43; Gap Junctions; Gene Expression; Ischemia; Marine Toxins; Myocytes, Cardiac; Okadaic Acid; Oxazoles; Phosphorylation; Polyenes; Pyrones; Rats; Rats, Sprague-Dawley

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.

Topics: Alkenes; Animals; Binding Sites; Calcineurin Inhibitors; Calcium; Cholecystokinin; Cyclic AMP; Cyclosporine; DNA-Binding Proteins; Enzyme Inhibitors; Marine Toxins; Okadaic Acid; Oxazoles; Pancreas; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Polyenes; Pyrones; Rats; Rats, Sprague-Dawley; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factors

Collagen and the cross-linked collagen-related peptide (CRP-XL) each induced platelet p38 mitogen-activated protein kinase (p38) phosphorylation after 2 min. Subsequent dephosphorylation occurred in platelets activated with collagen, but not with CRP-XL, demonstrating glycoprotein VI-independent regulation of p38. Okadaic acid and fostriecin, inhibitors specific for protein phosphatase 2A (PP2A), blocked p38 dephosphorylation, and PP2A co-immunoprecipitated with phospho-p38. In addition, use of phenylarsine oxide suggested that tyrosine phosphatases and PP2A may act in concert to dephosphorylate p38. Finally, regulation of p38 in collagen-stimulated Glanzmann's platelets was indistinguishable from that in normal platelets, showing that p38 regulation is independent of integrin alphaIIbbeta3.

Topics: Alkenes; Animals; Blood Platelets; Carrier Proteins; Cattle; Collagen; Enzyme Inhibitors; Humans; In Vitro Techniques; Kinetics; Mitogen-Activated Protein Kinases; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Peptides; Phosphoprotein Phosphatases; Phosphorylation; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Polyenes; Protein Phosphatase 2; Pyrones

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.

Topics: Alkenes; Antigens, Nuclear; Catalysis; Catalytic Domain; Cell Line; Cells, Cultured; DNA Damage; DNA Helicases; DNA Repair; DNA-Activated Protein Kinase; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Ku Autoantigen; Microcystins; Nuclear Proteins; Okadaic Acid; Peptides, Cyclic; Phosphates; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Precipitin Tests; Protein Binding; Protein Kinases; Protein Phosphatase 1; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Pyrones; Recombinant Proteins; Time Factors

Previous studies indicated that cells whose chromatin is naturally compacted at the time of radiation are hypersensitive to radiation-induced killing, primarily by single-hit inactivation. Some chemicals that are known to promote chromatin compaction in interphase cells are here investigated for their radiosensitizing potential.. Okadaic acid (OA), a protein phosphatase inhibitor, fostriecin (FC), a topoisomerase II inhibitor and trichostatin A (TSA), a histone deacetylase inhibitor, were reported to promote chromatin compaction in mammalian cells. Asynchronous populations of HT-29 (human colon carcinoma) cells were exposed to various concentrations of OA, FC and TSA for various times before irradiation with various doses of Cs-137 gamma-rays and toxicity and radiosensitization were measured. Induced chromatin compaction was visualized by electron microscopy (EM). Histone 1 (H1) and histone 3 (H3) phosphorylation was measured by Western blotting, whole-cell fluorescence microscopy and confocal microscopy.. OA and FC produced significant radiosensitization at 2 Gy after short (2 h) exposures. These chemical treatments also produced increased phosphorylation of H3 and increased chromatin compaction as measured by EM. A 2-h exposure of cells to TSA had no effect on cell radiosensitivity, histone phosphorylation or chromatin condensation. However, a 16-h exposure to TSA produced significant radiosensitization, histone phosphorylation and chromatin condensation, presumably by secondary mechanisms.. These data are consistent with the hypothesis that compacted chromatin is a hypersensitive target for radiation killing. Furthermore, the modulation of chromatin conformation by drugs selectively in tumour cells might radiosensitize tumours whose cells are intrinsically radioresistant.

Topics: Alkenes; Cell Survival; Chromatin; Histones; HT29 Cells; Humans; Hydroxamic Acids; Immunohistochemistry; Neoplasms; Okadaic Acid; Polyenes; Pyrones; Radiation Tolerance

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

Fostriecin, a structurally unique phosphate ester, is presently under evaluation in clinical trials to determine its potential use as an antitumor drug in humans. Fostriecin has been reported as having inhibitory activity against DNA topoisomerase type II and protein phosphatases implicated in cell-cycle control. However, the relative contribution of these mechanisms to the antitumor activity of fostriecin has not yet been elucidated. In this study, after confirming that fostriecin is a potent inhibitor of serine/threonine protein phosphatase type 2A and a weak inhibitor of serine/threonine protein phosphatase type 1, we show that fostriecin inhibits approximately 50% of the divalent cation independent serine/threonine protein phosphatase (PPase) activity contained in whole cell homogenates of Chinese hamster ovary cells at concentrations associated with antitumor activity (1-20 microM). Investigations into the cellular effects produced by fostriecin treatment reveal that 1-20 microM fostriecin induces a dose-dependent arrest of cell growth during the G2-M phase of the cell cycle. Immunostaining of treated cells indicates that growth arrest occurs before the completion of mitosis and that fostriecin-induced growth arrest is associated with the aberrant amplification of centrosomes, which results in the formation of abnormal mitotic spindles. The "mitotic block" induced by fostriecin is reversible if treatment is discontinued in <24 h. However, after approximately 24-30 h of continuous treatment, growth arrest is not reversible, and treated cells die even when placed in fostriecin-free media. Correlative studies conducted with established PPase inhibitors reveal that, when applied at concentrations that inhibit PPase activity to a comparable extent, both okadaic acid and cantharidin also induce aberrant centrosome replication, the appearance of multiple aberrant mitotic spindles, and G2-M-phase growth arrest. These studies add additional support to the concept that PPase inhibition underlies the antitumor activity of fostriecin and suggest that other type-selective PPase inhibitors should be evaluated for potential antitumor activity.

Topics: Alkenes; Animals; Antineoplastic Agents; Cantharidin; Centrosome; CHO Cells; Cricetinae; Enzyme Inhibitors; G2 Phase; Mitosis; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Pyrones; RNA, Messenger; Spindle Apparatus; Tumor Suppressor Protein p53

Fostriecin, an antitumor antibiotic produced by Streptomyces pulveraceus, is a strong inhibitor of type 2A (PP2A; IC50 3.2 nM) and a weak inhibitor of type 1 (PP1; IC50 131 microM) serine/threonine protein phosphatases. Fostriecin has no apparent effect on the activity of PP2B, and dose-inhibition studies conducted with whole cell homogenates indicate that fostriecin also inhibits the native forms of PP1 and PP2A. Studies with recombinant PP1/PP2A chimeras indicate that okadaic acid and fostriecin have different binding sites.

Topics: Alkenes; Animals; Antibiotics, Antineoplastic; Brain; Cattle; Enzyme Inhibitors; Isoenzymes; Kinetics; Muscle, Skeletal; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylase Kinase; Polyenes; Pyrones; Rabbits; Recombinant Fusion Proteins

DNA topoisomerase II (topo II) is required at mitosis in yeast for high chromosome condensation and for chromosome segregation. Recent studies on intact mammalian cells using topo II inhibitors that do not stabilize cleavable complexes also suggest a requirement for topo II for complete chromosome condensation and perhaps also for entry into mitosis. We have investigated the effects of merbarone, ICRF-187, and aclarubicin, three topo II inhibitors that do not stabilize the cleavable complex, on entry into mitosis and on chromosome condensation in BHK and in tsBN2 cells. We have compared their effects with those of etoposide, a topo II inhibitor that stabilizes the cleavable complex. All inhibitors induced a concentration-dependent G2 delay or arrest that could be overcome with fostriecin or okadaic acid or by inactivation of RCC1 in tsBN2 cells. Mitotic chromosomes from cells treated with etoposide were extensively fragmented, whereas mitotic chromosomes from cells treated with merbarone, ICRF-187, or aclarubicin were intact but elongated and tangled. These results provide strong evidence that topo II activity is required in chromosome condensation for final coiling of the chromatids. Our results also indicate that protein phosphatases and RCC1 play a role in G2 delay induced by all inhibitors, whether they do or do not stabilize the cleavable complex.

Topics: Aclarubicin; Alkenes; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Cell Cycle Proteins; Cell Line; Chromosomes; Cricetinae; DNA-Binding Proteins; Enzyme Inhibitors; Etoposide; G2 Phase; Guanine Nucleotide Exchange Factors; Kidney; Mitosis; Nuclear Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Pyrones; Razoxane; Temperature; Thiobarbiturates; Topoisomerase II Inhibitors

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.

Topics: Alkaloids; Alkenes; Animals; CDC2 Protein Kinase; Chromosomes; Cyclins; Ethers, Cyclic; G2 Phase; Genes, cdc; Histones; Mice; Mitosis; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Protamine Kinase; Protein Kinase C; Protein Phosphatase 1; Pyrones; Staurosporine; Temperature; Tumor Cells, Cultured