okadaic-acid and olomoucine

Expression of CYP1A1 mRNA in mouse hepatocytes in primary culture was investigated. The expression was obvious on day 3 of culture without addition of any known ligands of the aryl hydrocarbon receptor and increased with culture period. Removal of insulin from and addition of hydrogen peroxide to the medium enhanced and suppressed the expression, respectively. The CYP1A1 mRNA expression was also enhanced in the presence of anti-oxidant, t-butylhydroquinone, in the medium. Several kinds of kinase inhibitors markedly increased the CYP1A1 mRNA expression. In contrast, the inhibitory expression was prolonged in the presence of okadaic acid, a potent inhibitor of serine/threonine phosphatase PP1 and PP2. These observations suggest that there might be a repressive pathway in the regulation of CYP1A1 mRNA expression and that the presently observed expression pathway differs at several points from those previously reported, such as ligand-activated aryl hydrocarbon receptor- or omeprazole-mediated expression. Modulation of CYP1A2 mRNA expression after exposing hepatocytes to agents affecting phosphorylation pathways differed from that of CYP1A1 mRNA. This implies that regulatory pathways for CYP1A1 and CYP1A2 expression may differ.

Topics: Animals; Cells, Cultured; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Genistein; Hepatocytes; Hydrogen Peroxide; Hydroquinones; Insulin; Isoflavones; Kinetin; Mice; Mice, Inbred C57BL; Okadaic Acid; Phosphorylation; Purines; RNA, Messenger; Roscovitine; Time Factors; Transcriptional Activation

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane-bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule-dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin-driven motility in axons. This pathway involves enzymatic activities of cyclin-dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase-3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimer's disease are discussed.

Topics: Animals; Axonal Transport; Cell Movement; Cells, Cultured; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Decapodiformes; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Growth Cones; Immunoblotting; Immunohistochemistry; Kinesins; Kinetics; Kinetin; Models, Biological; Neurites; Neurons; Okadaic Acid; Phosphorylation; Protein Binding; Purines; Rats; Recombinant Proteins; Substrate Specificity

Many dramatic alterations in various cellular processes during the cell cycle are known to involve ion channels. In ascidian embryos and Caenorhabditis elegans oocytes, for example, the activity of inwardly rectifying Cl(-) channels is enhanced during the M phase of the cell cycle, but the mechanism underlying this change remains to be established. We show here that the volume-sensitive Cl(-) channel, ClC-2 is regulated by the M-phase-specific cyclin-dependent kinase, p34(cdc2)/cyclin B. ClC-2 channels were phosphorylated by p34(cdc2)/cyclin B in both in vitro and cell-free phosphorylation assays. ClC-2 phosphorylation was inhibited by olomoucine and abolished by a (632)Ser-to-Ala (S632A) mutation in the C-terminus, indicating that (632)Ser is a target of phosphorylation by p34(cdc2)/cyclin B. Injection of activated p34(cdc2)/cyclin B attenuated the ClC-2 currents but not the S632A mutant channel currents expressed in Xenopus oocytes. ClC-2 currents attenuated by p34(cdc2)/cyclin B were increased by application of the cyclin-dependent kinase inhibitor, olomoucine (100 microM), an effect that was inhibited by calyculin A (5 nM) but not by okadaic acid (5 nM). A yeast two-hybrid system revealed a direct interaction between the ClC-2 C-terminus and protein phosphatase 1. These data suggest that the ClC-2 channel is also counter-regulated by protein phosphatase 1. In addition, p34(cdc2)/cyclin B decreased the magnitude of ClC-2 channel activation caused by cell swelling. As the activities of both p34(cdc2)/cyclin B and protein phosphatase 1 vary during the cell cycle, as does cell volume, the ClC-2 channel could be regulated physiologically by these factors.

Topics: Animals; Chloride Channels; CLC-2 Chloride Channels; Cyclin-Dependent Kinases; Enzyme Inhibitors; Female; Kinetics; Kinetin; Marine Toxins; Mitosis; Okadaic Acid; Oocytes; Oxazoles; Phosphorylation; Purines; Recombinant Proteins; Xenopus

Organization of intermediate filament, a major component of cytoskeleton, is regulated by protein phosphorylation/dephosphorylation, which is a dynamic process governed by a balance between the activities of involved protein kinases and phosphatases. Blocking dephosphorylation by protein phosphatase inhibitors such as okadaic acid (OA) leads to an apparent activation of protein kinase(s) and to genuine activation of phosphatase-regulated protein kinase(s). Treatment of 9L rat brain tumor cells with OA results in a drastically increased phosphorylation of vimentin, an intermediate filament protein. In-gel renaturing assays and in vitro kinase assays using vimentin as the exogenous substrate indicate that certain protein kinase(s) is activated in OA-treated cells. With specific protein kinase inhibitors, we show the possible involvement of the cdc2 kinase- and p38 mitogen-activated protein kinase (p38MAPK)-mediated pathways in this process. Subsequent in vitro assays demonstrate that vimentin may serve as an excellent substrate for MAPK-activated protein kinase-2 (MAPKAPK-2), the downstream effector of p38MAPK, and that MAPKAPK-2 is activated with OA treatment. Comparative analysis of tryptic phosphopeptide maps also indicates that corresponding phosphopeptides emerged in vimentin from OA-treated cells and were phosphorylated by MAPKAPK-2. Taken together, the results clearly demonstrate that MAPKAPK-2 may function as a vimentin kinase in vitro and in vivo. These findings shed new light on the possible involvement of the p38MAPK signaling cascade, via MAPKAPK-2, in the maintenance of integrity and possible physiological regulation of intermediate filaments.

Topics: Animals; Brain Neoplasms; Cytosol; Electrophoresis, Gel, Two-Dimensional; Enzyme Activation; Gliosarcoma; Imidazoles; Intracellular Signaling Peptides and Proteins; Kinetin; Okadaic Acid; Peptide Mapping; Phosphorylation; Protein Serine-Threonine Kinases; Purines; Pyridines; Rats; Tumor Cells, Cultured

In Alzheimer's disease, tau protein becomes hyperphosporylated, which can contribute to neuronal degeneration. However, the implicated protein kinases are still unknown. Now we report that lithium (an inhibitor of glycogen synthase kinase-3) causes tau dephosphorylation at the sites recognized by antibodies Tau-1 and PHF-1 both in cultured neurons and in vivo in rat brain. This is consistent with a major role for glycogen synthase kinase-3 in modifying proline-directed sites on tau protein within living neurons under physiological conditions. Lithium also blocks the Alzheimer's disease-like proline-directed hyperphosphorylation of tau protein which is observed in neurons treated with a phosphatase inhibitor. These data raise the possibility of using lithium to prevent tau hyperphosphorylation in Alzheimer's disease.

Topics: Alzheimer Disease; Animals; Blotting, Western; Brain; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cyclin-Dependent Kinases; Enzyme Inhibitors; Flavonoids; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Humans; Kinetin; Lithium; Neurons; Okadaic Acid; Phosphorylation; Proline; Purines; Rats; tau Proteins

To examine whether microtubule dynamic instability can be rapidly regulated during interphase, we used video-enhanced differential interference contrast (DIC) microscopy to observe individual microtubules at the periphery of living newt lung epithelial cells. Microtubules were observed before and after perfusion with either the phosphatase inhibitor okadaic acid or the kinase inhibitors staurosporine or olomoucine. Addition of these inhibitors caused rapid changes in dynamic instability. Thirty to sixty seconds after perfusion with 0.2-1 microM okadaic acid, a 1.5-fold increase in elongation velocity and small increases in catastrophe and rescue frequencies were observed. In contrast, treatment with 40-200 nM staurosporine decreased microtubule elongation and shortening velocities approximately 2-fold, and catastrophes were slightly more frequent. Olomoucine, at 100 microM, had similar effects. Transition dynamics were further examined by probabilistic analysis, which showed that microtubules become more likely to undergo catastrophe as they elongated and more likely to undergo rescue as they shortened, an effect previously called microtubule "memory." This memory effect for catastrophes was observed in untreated and okadaic acid-treated cells but was abolished by staurosporine or olomoucine. In contrast, the memory effect for rescue was unaffected by these treatments, suggesting that catastrophe and rescue proceed via distinct, multistep mechanisms. Overall, these results demonstrate that microtubule assembly regulators can be altered rapidly by inhibition of either kinases or phosphatases and suggest that, in the absence of inhibitors, these regulators exist in a dynamic equilibrium between phosphorylated and dephosphorylated states.

Topics: Animals; Cells, Cultured; Enzyme Inhibitors; Epithelium; Kinetin; Lung; Microscopy, Interference; Microtubules; Okadaic Acid; Phosphorylases; Phosphotransferases; Purines; Salamandridae; Staurosporine; Time Factors

Histone H1 is highly phosphorylated in mitotic HeLa cells, but is quickly dephosphorylated in vivo at the end of mitosis and in vitro following cell lysis. We show here that okadaic acid and microcystin-LR block the in vitro dephosphorylation of H1 and that they do so directly by inhibiting the histone H1 phosphatase rather than by some indirect mechanism. The concentrations of microcystin and okadaic acid required for inhibition strongly suggest that the histone H1 phosphatase is either PP1 or an unknown protein phosphatase with okadaic acid-sensitivity similar to PP1. The histone H1 phosphatase is predominantly located in chromosomes with at most one copy for every 86 nucleosomes. This tends to support its identification as PP1, since localization in mitotic chromosomes is a characteristic of PP1 but not of the other known okadaic acid-sensitive protein phosphatases. We also show that treatment of metaphase-arrested HeLa cells with staurosporine and olomoucine, inhibitors of p34cdc2 and other protein kinases, rapidly induces reassembly of interphase nuclei and dephosphorylation of histone H1 without chromosome segregation. This result indicates that protein kinase activity must remain elevated to maintain a mitotic block. Using this as a model system for the M- to G1-phase transition, we present evidence from inhibitor studies suggesting that the in vivo histone H1 phosphatase may be either PP1 or another phosphatase with similar okadaic acid-sensitivity, but not PP2A.

Topics: Cell Nucleus; Chromatin; Enzyme Inhibitors; HeLa Cells; Histones; Humans; Kinetin; Marine Toxins; Metaphase; Microcystins; Mitosis; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 1; Protein Phosphatase 2; Purines; Staurosporine