okadaic-acid and endothall

The major excitatory neurotransmitter Glutamate acts on both ionotropic and metabotropic glutamate receptors (mGluRs) in the central nervous system. mGluR5, a member of the group I mGluR family is widely expressed throughout the brain and plays important roles in a variety of neuronal processes including various forms of synaptic plasticity. This receptor is also involved in various neuropsychiatric disorders, viz., Fragile X syndrome, autism etc. It has been reported that mGluR5 undergoes desensitization and subsequently internalization on ligand exposure in various cell types. However, the downstream events after the internalization and the molecular players involved in the post-endocytic events of this receptor have not been studied. In the present study, we find that subsequent to internalization mGluR5 enters the recycling compartment. After that the receptor recycles back to the cell surface. We also show here that the recycling of mGluR5 is dependent on protein phosphatases. Our data suggest that mGluR5 recycling is completely dependent on the activity of PP2A whereas, PP2B has partial effect on this process. Thus our study suggests that mGluR5 recycles back to the cell surface after ligand-dependent internalization and protein phosphatases that have been implicated in various forms of synaptic plasticity have differential effects on the recycling of mGluR5.

Topics: Animals; Dicarboxylic Acids; Endocytosis; HEK293 Cells; Hippocampus; Humans; Mice, Inbred C57BL; Neurons; Okadaic Acid; Protein Phosphatase 2; Receptor, Metabotropic Glutamate 5

Protein phosphatase 5 (PP5), a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects, Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa), 490 (55.82 kDa) and 491 (56.07 kDa) amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat) domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cantharidin; Cloning, Molecular; Cluster Analysis; Computational Biology; Dicarboxylic Acids; DNA Primers; DNA, Complementary; Escherichia coli; Gene Expression Regulation, Enzymologic; Kinetics; Molecular Sequence Data; Moths; Nuclear Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phylogeny; Recombinant Proteins; Sequence Analysis, DNA; Species Specificity

Profilin-1 (PFN1) plays an important role in the control of actin dynamics, and could represent an important therapeutic target in several diseases. We previously identified PFN1 as a huntingtin aggregation inhibitor, and others have implicated it as a tumor-suppressor. Rho-associated kinase (ROCK) directly phosphorylates PFN1 at Ser-137 to prevent its binding to polyproline sequences. This negatively regulates its anti-aggregation activity. However, the phosphatase that dephosphorylates PFN1 at Ser-137, and thus activates it, is unknown. Using a phospho-specific antibody against Ser-137 of PFN1, we characterized PFN1 dephosphorylation in cultured cells based on immunocytochemistry and a quantitative plate reader-based assay. Both okadaic acid and endothall increased pS137-PFN1 levels at concentrations more consistent with their known IC(50)s for protein phosphatase 1 (PP1) than protein phosphatase 2A (PP2A). Knockdown of the catalytic subunit of PP1 (PP1Cα), but not PP2A (PP2ACα), increased pS137-PFN1 levels. PP1Cα binds PFN1 in cultured cells, and this interaction was increased by a phosphomimetic mutation of PFN1 at Ser-137 (S137D). Together, these data define PP1 as the principal phosphatase for Ser-137 of PFN1, and provide mechanistic insights into PFN1 regulation by phosphorylation.

Topics: Animals; Blotting, Western; Dicarboxylic Acids; Dose-Response Relationship, Drug; HEK293 Cells; HeLa Cells; Humans; Immunohistochemistry; Mice; NIH 3T3 Cells; Okadaic Acid; Phosphorylation; Profilins; Protein Binding; Protein Phosphatase 1; Protein Phosphatase 2; RNA Interference; Serine

The beneficial effect of phosphodiesterase 5A inhibition in ischemia/reperfusion injury and cardiac hypertrophy is well established. Inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1) exerts beneficial effects on these same conditions, and a possible link between these therapeutic strategies was suggested. Experiments were performed in isolated cat cardiomyocytes to gain insight into the intracellular pathway involved in the reduction of NHE-1 activity by phosphodiesterase 5A inhibition. NHE-1 activity was assessed by the rate of intracellular pH recovery from a sustained acidic load in the absence of bicarbonate. Phosphodiesterase 5A inhibition with sildenafil (1 μmol/L) did not affect basal intracellular pH; yet, it did decrease proton efflux (J(H); in millimoles per liter per minute) after the acidic load (proton efflux: 6.97±0.43 in control versus 3.31±0.58 with sildenafil; P<0.05). The blockade of both protein phosphatase 1 and 2A with 100 nmol/L of okadaic acid reverted the sildenafil effect (proton efflux: 6.77±0.82). In contrast, selective inhibition of protein phosphatase 2A (1 nmol/L of okadaic acid or 100 μmol/L of endothall) did not (3.86±1.0 and 2.61±1.2), suggesting that only protein phosphatase 1 was involved in sildenafil-induced NHE-1 inhibition. Moreover, sildenafil prevented the acidosis-induced increase in NHE-1 phosphorylation without affecting activation of the extracellular signal-regulated kinase 1/2-p90(RSK) pathway. Our results suggest that phosphodiesterase 5A inhibition decreases NHE-1 activity, during intracellular pH recovery after an acidic load, by a protein phosphatase 1-dependent reduction in NHE-1 phosphorylation.

Topics: Animals; Biological Transport; Cats; Cells, Cultured; Cyclic Nucleotide Phosphodiesterases, Type 5; Dicarboxylic Acids; Enzyme Inhibitors; Hydrogen-Ion Concentration; Immunoblotting; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Okadaic Acid; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Phosphorylation; Piperazines; Protein Phosphatase 1; Protons; Purines; Ribosomal Protein S6 Kinases, 90-kDa; Sildenafil Citrate; Sodium-Hydrogen Exchangers; Sulfones

This study aimed to identify the signaling pathway for the proposed link between phosphodiesterase-5A (PDE5A) inhibition and decreased cardiac Na(+)/H(+) exchanger (NHE-1) activity.. NHE-1 activity was assessed in rat isolated papillary muscles by the Na(+)-dependent initial pH(i) recovery from a sustained acidosis (ammonium prepulse). ERK1/2, p90RSK and NHE-1 phosphorylation state during acidosis was determined.. PDE5A inhibition (1 μmol/L sildenafil, SIL) did not modify basal pH(i) but significantly blunted pH(i) recovery after sustained acidosis. Although preventing ERK1/2- p90RSK signaling pathway (10 μmol/L U0126) mimicked SIL effect, SIL did not blunt the acidosis-mediated increase in kinases activation. SIL+U0126 did not show additive effect on NHE-1 activity. Then, we hypothesized that SIL could be activating phophasatases (PP1 and/or PP2A) to directly dephosphorylate NHE-1 despite preserved ERK1/2-p90RSK activation. Non-specific phosphatases inhibition (1 μmol/L okadaic acid) canceled SIL effect on pH(i) recovery from acidosis. Same result was observed by inhibiting PP2A either with a lower dose of okadaic acid (1 nmol/L) or, more specifically, with 100 μmol/L endothall. Consistently, NHE-1 phosphorylation at Ser703 increased after acidosis, SIL prevented this effect and PP2A inhibition (endothall) reverted SIL effect.. We suggest that PDE5A inhibitors decrease NHE-1 phosphorylation and activity through a mechanism that involves PP2A activation.

Topics: Acidosis; Animals; Butadienes; Cyclic Nucleotide Phosphodiesterases, Type 5; Dicarboxylic Acids; Hydrogen-Ion Concentration; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Okadaic Acid; Papillary Muscles; Phosphodiesterase Inhibitors; Phosphorylation; Piperazines; Protein Phosphatase 1; Protein Phosphatase 2; Purines; Rats; Ribosomal Protein S6 Kinases, 90-kDa; Sildenafil Citrate; Sodium-Hydrogen Exchangers; Sulfones

It is becoming increasingly clear that protein phosphatases are important modulators of cellular function and that disruption of these proteins are involved in neurodegenerative disease processes. Serine/threonine protein phosphatases (PP) such as protein phosphatase PP1, PP2A, and calcineurin are involved in hyperphosphorylation of tau- as well as beta-amyloid-induced cell death. We have previously shown serine/threonine protein phosphatases to be involved in estrogen-mediated neuroprotection. The purpose of this study was to delineate the role of PP1, PP2A, and calcineurin in the mechanism of estrogen mediated neuroprotection against oxidative stress and excitotoxicity. Treatment with protein phosphatases inhibitor II, endothall, or cyclosporin A, which are specific inhibitors of PP1, PP2A, and calcineurin, respectively, did not have an effect on cell viability. However, in combination, these inhibitors adversely affected cell survival, which suggests the importance of serine/threonine protein phosphatases in maintenance of cellular function. Inhibitors of PP1, PP2A, and calcineurin attenuated the protective effects of estrogen against glutamate-induced -neurotoxicity but did not completely abrogate the estrogen-mediated protection. The attenuation of estrogen-induced neuroprotection was achieved through decrease in the activity of theses serine/threonine phosphatases without the concomitant decrease in protein expression. In an animal model, transient middle cerebral artery occlusion caused a 50% decrease in levels of PP1, PP2A, and PP2B ipsilateral to the lesion in a manner that was prevented by estradiol pretreatment. Therefore, we conclude that in the face of cytotoxic challenges in vitro and in vivo, estrogens maintain the function of PP1, PP2A, and calcineurin.

Topics: Animals; Brain Neoplasms; Calcineurin; Calcineurin Inhibitors; Cell Line, Tumor; Cell Survival; Cyclosporine; Dicarboxylic Acids; Drug Interactions; Enzyme Inhibitors; Estrogens; Glioma; Glutamic Acid; Mice; Neurons; Neuroprotective Agents; Neurotoxins; Okadaic Acid; Oxidative Stress; Protein Phosphatase 1; Protein Phosphatase 2; Rats; Stroke

A novel rice (Oryza sativa L.) gene, homologous to a sorghum pathogenesis-related class 10 protein gene, was cloned from a cDNA library prepared from 2-week-old jasmonic acid-treated rice seedling leaves, and named as JIOsPR10 (jasmonate inducible). JIOsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17,173.23 Da and a pI of 5.84. JIOsPR10 was highly similar (77%) to the sorghum PR10 protein, but showed less than 55% similarity with other identified PR10s at the amino acid level. Genomic Southern analyses indicated the presence of related genes in the rice genome. The JIOsPR10 transcript was not detected in the healthy leaves, and was not induced after cut. Further expression analysis revealed that the signaling components of defense/stress pathways, jasmonate, salicylate, and H(2)O(2) significantly up-regulated the JIOsPR10 mRNA over the cut control, whereas two other stress regulators, ethylene and abscisic acid, failed to induce its expression. Interestingly the protein phosphatase (PP) inhibitors, cantharidin, endothall, and okadaic acid, rapidly and potently up-regulated the JIOsPR10 expression, suggesting involvement of the phosphorylation/dephosphorylation events. Additionally, the inducible expression of the JIOsPR10 gene was influenced by light signal(s). Finally, the blast pathogen (Magnaporthe grisea) also specifically elicited the accumulation of JIOsPR10 mRNA in leaves. Induction of the JIOsPR10 gene expression by signaling molecules, PP inhibitors and pathogen attack, strongly indicate a role for this novel gene in rice self-defense/stress response(s).

Topics: Amino Acid Sequence; Base Sequence; Blotting, Northern; Blotting, Southern; Cantharidin; Cloning, Molecular; Cyclopentanes; Dicarboxylic Acids; DNA, Complementary; Enzyme Inhibitors; Gene Library; Genome, Plant; Hydrogen Peroxide; Molecular Sequence Data; Okadaic Acid; Oryza; Oxylipins; Peptides; Phosphoprotein Phosphatases; Phosphorylation; Phylogeny; Plant Leaves; Plant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salicylic Acid; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Signal Transduction; Time Factors; Up-Regulation

The catalytic subunit of protein phosphatase 2A (PP2Ac) was purified from Neurospora crassa extract by (NH4)2SO4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M(r) of PP2Ac was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2Ac was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2Ac is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2Ac did not cross-react with N. crassa PP2Ac, indicating sequence differences outside the catalytic core of the enzyme.

Topics: Ammonium Sulfate; Animals; Cantharidin; Chemical Precipitation; Chromatography; Dicarboxylic Acids; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Ethanol; Ethers, Cyclic; Humans; Marine Toxins; Microcystins; Molecular Weight; Muscle, Skeletal; Neurospora crassa; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 2; Rabbits; Sodium Fluoride

The toxic effects of cantharidin from blister beetles and its analogs, including the herbicide endothall, are attributable to their high affinity and specificity for a cantharidin-binding protein (CBP). An ammonium sulfate precipitate of mouse liver cytosol was purified by five chromatographic steps to isolate CBP in 14% yield and > 99% purity as monitored by [3H]cantharidin-binding activity. The purification factor of 2230-fold corresponds to a CBP content of 0.045% of the liver cytosolic protein. CBP is a heterodimer consisting of a 61-kDa alpha subunit and a 39-kDa beta subunit. Amino acid sequences of four peptides from CBP-alpha and three peptides from CBP-beta are identical with deduced amino acid sequences for the A alpha regulatory and C beta catalytic subunits, respectively, of protein phosphatase 2A (PP2A). This assignment of CBP as PP2A-AC from structural evidence is supported by biochemical studies with selective substrates and inhibitors. CBP dephosphorylation of phosphorylase alpha is sensitive not only to okadaic acid, as with PP2A, but also to cantharidin and its analogs, consistent with their potency in blocking the radioligand binding site of CBP. Okadaic acid is a potent inhibitor of [3H]cantharidin binding to CBP. PP2A is present in many mammalian tissues and in plants and is involved in regulatory phosphorylation-dephosphorylation events which modulate multiple cellular functions. Inhibition of PP2A activity may account for the diverse effects and toxicity of cantharidin and its analogs, including the herbicide endothall, in mammals and possibly plants.

Topics: Amino Acid Sequence; Animals; Cantharidin; Carrier Proteins; Cytosol; Dicarboxylic Acids; Ethers, Cyclic; Liver; Mice; Molecular Sequence Data; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; Sequence Alignment