okadaic-acid and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP), which represses and reversibly regulates the translation of a subset of mRNAs in dendrites. Protein synthesis can be rapidly stimulated by mGluR-induced and protein phosphatase 2a (PP2A)-mediated dephosphorylation of FMRP, which is coupled to the dissociation of FMRP and target mRNAs from miRNA-induced silencing complexes. Here, we report the rapid ubiquitination and ubiquitin proteasome system (UPS)-mediated degradation of FMRP in dendrites upon DHPG (3,5-dihydroxyphenylglycine) stimulation in cultured rat neurons. Using inhibitors to PP2A and FMRP phosphomutants, degradation of FMRP was observed to depend on its prior dephosphorylation. Translational induction of an FMRP target, postsynaptic density-95 mRNA, required both PP2A and UPS. Thus, control of FMRP levels at the synapse by dephosphorylation-induced and UPS-mediated degradation provides a mode to regulate protein synthesis.

Topics: Analysis of Variance; Animals; Boronic Acids; Bortezomib; Cells, Cultured; Dendrites; Disks Large Homolog 4 Protein; Drosophila Proteins; Embryo, Mammalian; Enzyme Inhibitors; Female; Fragile X Mental Retardation Protein; Gene Expression Regulation; Green Fluorescent Proteins; Hippocampus; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Leupeptins; Male; Membrane Proteins; Methoxyhydroxyphenylglycol; Mutation; Neurons; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Biosynthesis; Pyrazines; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; RNA, Messenger; Serine; Signal Transduction; Synapses; Transfection; Ubiquitination

The chromosomal passenger complex (CPC) senses tension defects at the kinetochore to activate the spindle assembly checkpoint, and helps to position the cleavage furrow. The CPC, consisting of INCENP, Survivin, Borealin and Aurora B localizes to the inner centromere at metaphase and re-localizes to the spindle midzone at anaphase; several CPC functions are regulated by post-translational modification. Borealin is phosphorylated at multiple sites and phosphorylation at S219 causes Borealin to migrate more slowly upon electrophoresis. Here we find that Cdk1 can induce a mobility shift of Borealin, suggesting that S219 phosphorylation is under Cdk1 control. However, Cdk1 is inefficient at phosphorylating purified Borealin in vitro. A yeast orthologue of Borealin, Npl1, is dephosphorylated by the phosphatase Cdc14. We find no difference in the mobility shift of Borealin in human cells lacking either Cdc14A or Cdc14B. In contrast, the phosphatase inhibitor okadaic acid does delay the dephosphorylation of Borealin as cells exit mitosis. The proteasome inhibitor MG132 reduces Borealin phosphorylation in mitosis and increases the steady-state level of Borealin, especially in mutants lacking the C-terminus. However, a second, structurally unrelated proteasome inhibitor, lactacystin did not up-regulate Borealin. These results suggest that the effect of MG132 on Borealin is due to the inhibition of an intracellular protease other than the proteasome.

Topics: Acetylcysteine; Amino Acid Substitution; CDC2 Protein Kinase; Cell Cycle Proteins; Dual-Specificity Phosphatases; Gene Expression; HeLa Cells; Humans; Leupeptins; Okadaic Acid; Peptide Fragments; Phosphoric Monoester Hydrolases; Phosphorylation; Proteasome Inhibitors; Protein Processing, Post-Translational; Protein Transport; Proteolysis; Ubiquitin

The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCF(TIR1/AFB) (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H(+)-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H(+)-ATPase activation has yet to be elucidated. Here, we present direct evidence for H(+)-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H(+)-ATPase and increased H(+)-ATPase activity without altering the amount of the enzyme. Changes in both the phosphorylation level of H(+)-ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H(+)-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H(+)-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H(+)-ATPase phosphorylation. Our results define the activation mechanism of H(+)-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Membrane; Enzyme Activation; F-Box Proteins; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Hypocotyl; Indoleacetic Acids; Leupeptins; Marine Toxins; Okadaic Acid; Oxazoles; Phosphorylation; Proteasome Endopeptidase Complex; Proton-Translocating ATPases; Receptors, Cell Surface; Signal Transduction; Threonine; Time Factors; Transcription, Genetic

α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Cell Line, Tumor; Cerebral Cortex; Cycloheximide; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Lewy Bodies; Neurons; Okadaic Acid; Parkinson Disease; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Phosphatase 1; Protein Phosphatase 2; Protein Synthesis Inhibitors; Rabbits; Ubiquitin

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamines including dopamine. The relationship between proteasomal dysfunction and the etiology of Parkinson's disease has been suggested, but it is unknown if TH protein is affected by proteasomal dysfunctions. Here, we examined the effect of inhibition of ubiquitin-proteasomal pathway on biochemical characteristics of TH protein in the neuronal cells. Inhibition of 20S or 26S proteasome by proteasome inhibitor I, or MG-132 in NGF-differentiated PC12D cells induced dot-like immunoreactivities with the anti-(40)Ser-phosphorylated TH (p40-TH) antibody. These dots were tightly co-localized with ubiquitin and positive to Thioflavine-S staining. These dot-like immunoreactivities were not obvious when immunostaining was performed against total-TH or choline acetyltransferase. Western blotting analysis showed time-dependent increase of p40-TH in the Triton-insoluble fractions. We also examined the effect of okadaic acid, an inhibitor of protein phosphatase 2A, which is a phosphatase acting on p40-TH. Okadaic acid increased the amount of insoluble p40-TH. These data suggest that p40-TH is prone to be insolubilized and aggregated by dysfunction of an ubiquitin-proteasome system in PC12D cells.

Topics: Animals; Choline O-Acetyltransferase; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Leupeptins; Neurons; Okadaic Acid; Oligopeptides; PC12 Cells; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Multimerization; Protein Phosphatase 2; Rats; Time Factors; Tyrosine 3-Monooxygenase; Ubiquitin

In mammals, matured oocytes are arrested at the MII stage until fertilization, which is regulated by cytostaticfactor (CSF) activity. Maturation-promoting factor (MPF) and the mitogen-activated protein kinase (MAPK) pathway are known as candidates for CSF. Despite of the results that nuclear and perinuclear materials were dispensable for activation of MPF and MAPK in other species, our previous study in rats demonstrated that MPF activity was rapidly decreased after enucleation. We showed here for the first time that nuclear and perinuclear materials were indispensable for CSF activity in matured rat oocytes. In both cytoplasm-removed and enucleated oocytes, high activity of p34(cdc2) kinase was observed immediately after manipulation, but the activity of enucleated oocytes was dramatically reduced within 1 h. Cyclin B level was also decreased, corresponding with inactivation of p34(cdc2) kinase. In enucleated oocytes, the Mos level was dramatically decreased, and both MEK and MAPK dephosphorylation were also induced. A combined treatment with a proteasome inhibitor, MG132, and a protein phosphatase inhibitor, okadaic acid, dramatically improved both levels of p-MAPK and cyclin B in these enucleated oocytes. These data suggest that nuclear and perinuclear materials of matured rat oocytes suppress proteasome and protein phosphatase activation, which is indispensable for stability of CSF.

Topics: Animals; CDC2 Protein Kinase; Cell Nucleus; Cyclin B; Cytoplasm; Enzyme Activation; Female; Fetal Proteins; Genes, mos; In Vitro Techniques; Leupeptins; MAP Kinase Signaling System; Okadaic Acid; Oocytes; Phosphoprotein Phosphatases; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats

In a recent study, we demonstrated that vagal stimulation increases the survival of rats with myocardial infarction by inhibiting lethal arrhythmia through regulation of connexin43 (Cx43). However, the precise mechanisms for this effect remain to be elucidated. To investigate these mechanisms and the signal transduction for gap junction regulation, we investigated the effect of acetylcholine (ACh), a parasympathetic nerve system neurotransmitter, on the gap junction component Cx43 using H9c2 cells. When cells were subjected to hypoxia, the total Cx43 protein level was decreased. In contrast, pretreatment with ACh inhibited this effect. To investigate the signal transduction, cells were pretreated with L-NAME, a nitric oxide synthase inhibitor, followed by ACh and hypoxia. L-NAME was found to suppress the ACh effect. However, a NO donor, SNAP, partially inhibited the hypoxia-induced reduction in Cx43. To delineate the mechanisms of the decrease in Cx43 under hypoxia, cells were pretreated with MG132, a proteasome inhibitor. Proteasome inhibition produced a striking recovery of the decrease in the total Cx43 protein level under hypoxia. However, cotreatment with MG132 and ACh did not produce any further increase in the total Cx43 protein level. Functional studies using ACh or okadaic acid, a phosphatase inhibitor, revealed that both reagents inhibited the decrease in the dye transfer induced by hypoxia. These results suggest that ACh is responsible for restoring the decrease in the Cx43 protein level, resulting in functional activation of gap junctions.

Topics: Acetylcholine; Animals; Cell Communication; Cell Hypoxia; Cell Line; Connexin 43; Cysteine Proteinase Inhibitors; Gap Junctions; Leupeptins; Myocytes, Cardiac; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; S-Nitroso-N-Acetylpenicillamine; Time Factors

Tau-positive inclusions in oligodendrocytes are consistent neuropathological features of corticobasal degeneration, progressive supranuclear palsy, and frontotemporal dementias with Parkinsonism linked to chromosome 17. Here we show by immunohistochemistry that tau-positive oligodendroglial inclusion bodies also contain the small heat-shock protein (HSP) alphaB-crystallin but not HSP70. To study the molecular mechanisms underlying inclusion body formation, we engineered an oligodendroglia cell line (OLN-t40) to overexpress the longest human tau isoform. Treatment of OLN-t40 cells with okadaic acid (OA), an inhibitor of protein phosphatase 2A, caused tau hyperphosphorylation and a decrease in the binding of tau to microtubules. Simultaneously, tau-positive aggregates that also stained with the amyloid-binding dye thioflavin-S as well as with antibodies to tau and alphaB-crystallin were detected. However, they were only transiently expressed and were degraded within 24 hr. When the proteasomal apparatus was inhibited by carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG-132) after OA treatment, the aggregates were stabilized and were still detectable after 18 hr in the absence of OA. Incubation with MG-132 alone inhibited tau proteolysis and led to the induction of HSPs, including alphaB-crystallin and to its translocation to the perinuclear region, but did not induce the formation of thioflavin-S-positive aggregates. Hence, although tau hyperphosphorylation induced by protein phosphatase inhibition contributes to pathological aggregate formation, only hyperphosporylation of tau followed by proteasome inhibition leads to stable fibrillary deposits of tau similar to those observed in neurodegenerative diseases.

Topics: alpha-Crystallin B Chain; Benzothiazoles; Brain; Cell Line; Cysteine Endopeptidases; Cytoskeleton; Enzyme Inhibitors; Fluorescent Dyes; Humans; Immunohistochemistry; Inclusion Bodies; Leupeptins; Microtubules; Multienzyme Complexes; Okadaic Acid; Oligodendroglia; Phosphoprotein Phosphatases; Phosphorylation; Proteasome Endopeptidase Complex; Protein Phosphatase 2; tau Proteins; Tauopathies; Thiazoles

Activation of NF-kappa B by various cellular stimuli involves the phosphorylation and subsequent degradation of its inhibitor, I kappa B alpha, although the underlying mechanism remains unclear. In the present study, the role of serine/threonine phosphatases in the regulation of I kappa B alpha phosphorylation was investigated. Our studies demonstrate that incubation of human T cells with low concentrations (approximately 1-5 nM) of calyculin A or okadaic acid, potent inhibitors of protein phosphatase type 1 (PP-1) and type 2A (PP-2A), induces the phosphorylation of I kappa B alpha even in the absence of any cellular stimulus. This action of the phosphatase inhibitors, which is associated with the activation of the RelA.p50 NF-kappa B heterodimer, is not affected by agents that block the induction of I kappa B alpha phosphorylation by tumor necrosis factor alpha (TNF-alpha). Furthermore, the phosphorylated I kappa B alpha from calyculin A-treated cells, but not that from TNF-alpha-stimulated cells, is sensitive to PP-2A in vitro, suggesting the existence of fundamental differences in the phosphorylation of I kappa B alpha induced by the two different NF-kappa B inducers. However, induction of I kappa B alpha phosphorylation by both TNF-alpha and the phosphatase inhibitors is associated with the subsequent degradation of I kappa B alpha. We further demonstrate that TNF-alpha- and calyculin A-induced I kappa B alpha degradation exhibits similar but not identical sensitivities to a proteasome inhibitor. Together, these results suggest that phosphorylation of I kappa B alpha, mediated through both the TNF-alpha-inducible and the PP-2A-opposing kinases, may serve to target I kappa B alpha for proteasome-mediated degradation.

Topics: Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Drug Synergism; Enzyme Inhibitors; Ethers, Cyclic; Humans; I-kappa B Proteins; Leupeptins; Marine Toxins; Multienzyme Complexes; NF-kappa B; NF-KappaB Inhibitor alpha; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Proteasome Endopeptidase Complex; Protein Phosphatase 2; Protein Processing, Post-Translational; Signal Transduction; T-Lymphocytes; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha