Okadaic acid induces dephosphorylation of histone H1 in metaphase-arrested HeLa cells

1994 ◽  
Vol 107 (1) ◽  
pp. 267-273 ◽  
Author(s):  
J.R. Paulson ◽  
W.A. Ciesielski ◽  
B.R. Schram ◽  
P.W. Mesner
Keyword(s):  
Acid Concentration ◽  
Okadaic Acid ◽  
Hela Cells ◽  
Protein Phosphatase ◽  
Histone H1 ◽  
Protein Phosphatase 1 ◽  
Calyculin A ◽  
Phosphatase 2A ◽  
20 Nm ◽  
Normal Mitosis

It is shown here that treatment of metaphase-arrested HeLa cells with okadaic acid (0.15-2.5 microM) leads to dephosphorylation of histone H1. This effect is presumably due to the specific ability of okadaic acid to inhibit protein phosphatases 1 and/or 2A, because okadaic acid tetraacetate, which is not a phosphatase inhibitor, has no effect. Dephosphorylation of H1 does not occur if okadaic acid-treated cells are simultaneously treated with 20 nM calyculin A, or if the okadaic acid concentration is 5.0 microM or greater. The mechanism behind this phenomenon is not known. However, the results suggest that the chain of events leading to histone dephosphorylation may be negatively controlled by a protein phosphatase 2A, while the phosphatase which actually dephosphorylates H1 could be a protein phosphatase 1. It remains to be determined whether the phosphatase involved here is the same enzyme as that which dephosphorylates H1 at the end of normal mitosis.

scholarly journals Identification of Novel Serine/Threonine Protein Phosphatases inTrypanosoma cruzi: a Potential Role in Control of Cytokinesis and Morphology

2000 ◽  
Vol 68 (3) ◽  
pp. 1350-1358 ◽  
Author(s):  
George A. Orr ◽  
Craig Werner ◽  
Jun Xu ◽  
Marcia Bennett ◽  
Louis M. Weiss ◽  
...  
Keyword(s):  
Cell Division ◽  
Okadaic Acid ◽  
Blot Analysis ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Growth Arrest ◽  
Human Protein ◽  
Protein Phosphatase 1 ◽  
Calyculin A ◽  
Metacyclic Trypomastigote

ABSTRACT We cloned two novel Trypanosoma cruzi proteins by using degenerate oligonucleotide primers prepared against conserved domains in mammalian serine/threonine protein phosphatases 1, 2A, and 2B. The isolated genes encoded proteins of 323 and 330 amino acids, respectively, that were more homologous to the catalytic subunit of human protein phosphatase 1 than to those of human protein phosphatase 2A or 2B. The proteins encoded by these genes have been tentatively designated TcPP1α and TcPP1β. Northern blot analysis revealed the presence of a major 2.3-kb mRNA transcript hybridizing to each gene in both the epimastigote and metacyclic trypomastigote developmental stages. Southern blot analysis suggests that each protein phosphatase 1 gene is present as a single copy in the T. cruzi genome. The complete coding region for TcPP1β was expressed inEscherichia coli by using a vector, pTACTAC, with thetrp-lac hybrid promoter. The recombinant protein from the TcPP1β construct displayed phosphatase activity toward phosphorylasea, and this activity was preferentially inhibited by calyculin A (50% inhibitory concentration [IC50], ∼2 nM) over okadaic acid (IC50, ∼100 nM). Calyculin A, but not okadaic acid, had profound effects on the in vitro replication and morphology of T. cruzi epimastigotes. Low concentrations of calyculin A (1 to 10 nM) caused growth arrest. Electron microscopic studies of the calyculin A-treated epimastigotes revealed that the organisms underwent duplication of organelles, including the flagellum, kinetoplast, and nucleus, but were incapable of completing cell division. At concentrations higher than 10 nM, or upon prolonged incubation at lower concentrations, the epimastigotes lost their characteristic elongated spindle shape and had a more rounded morphology. Okadaic acid at concentrations up to 1 μM did not result in growth arrest or morphological alterations to T. cruziepimastigotes. Calyculin A, but not okadaic acid, was also a potent inhibitor of the dephosphorylation of 32P-labeled phosphorylase a by T. cruzi epimastigotes and metacyclic trypomastigote extracts. These inhibitor studies suggest that in T. cruzi, type 1 protein phosphatases are important for the completion of cell division and for the maintenance of cell shape.

Propofol Inhibits Phosphorylation of N -methyl-d-aspartate Receptor NR1 Subunits in Neurons

2006 ◽  
Vol 104 (4) ◽  
pp. 763-769 ◽  
Author(s):  
Seth Kingston ◽  
Limin Mao ◽  
Lu Yang ◽  
Anish Arora ◽  
Eugene E. Fibuch ◽  
...  
Keyword(s):  
Glutamate Receptors ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cortical Neurons ◽  
Protein Phosphatase 2A ◽  
Ionotropic Glutamate Receptors ◽  
Dependent Manner ◽  
Calyculin A ◽  
General Anesthetic ◽  
Phosphatase 2A

Background Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-D-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation. Methods The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons. Results Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits. Conclusions Together, these data indicate an inhibitory effect of a general anesthetic propofol on NMDAR NR1 subunit phosphorylation in neurons. This inhibition was mediated through a signaling mechanism involving activation of protein phosphatase 2A.

scholarly journals Regulation of the Cyclin B Degradation System by an Inhibitor of Mitotic Proteolysis

1998 ◽  
Vol 9 (7) ◽  
pp. 1817-1831 ◽  
Author(s):  
Elisabeth Vorlaufer ◽  
Jan-Michael Peters
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cyclin B ◽  
Protein Phosphatase 1 ◽  
Anaphase Promoting Complex ◽  
Egg Extracts ◽  
Mitotic Cyclin ◽  
Phosphatase 2A ◽  
Cytostatic Factor ◽  
Xenopus Egg Extracts

The initiation of anaphase and exit from mitosis depend on the anaphase-promoting complex (APC), which mediates the ubiquitin-dependent proteolysis of anaphase-inhibiting proteins and mitotic cyclins. We have analyzed whether protein phosphatases are required for mitotic APC activation. In Xenopus egg extracts APC activation occurs normally in the presence of protein phosphatase 1 inhibitors, suggesting that the anaphase defects caused by protein phosphatase 1 mutation in several organisms are not due to a failure to activate the APC. Contrary to this, the initiation of mitotic cyclin B proteolysis is prevented by inhibitors of protein phosphatase 2A such as okadaic acid. Okadaic acid induces an activity that inhibits cyclin B ubiquitination. We refer to this activity as inhibitor of mitotic proteolysis because it also prevents the degradation of other APC substrates. A similar activity exists in extracts of Xenopus eggs that are arrested at the second meiotic metaphase by the cytostatic factor activity of the protein kinase mos. In Xenopus eggs, the initiation of anaphase II may therefore be prevented by an inhibitor of APC-dependent ubiquitination.

Effect of Inhibitors of Protein Phosphatase 1 and 2A on Erythroid Colony Formation: An Investigation of the Specificities of Inhibitors

2001 ◽  
Vol 29 (2) ◽  
pp. 114-118 ◽  
Author(s):  
W Zhang ◽  
J Tamura ◽  
M Sakuraya ◽  
T Naruse ◽  
K Kubota
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Colony Formation ◽  
Protein Phosphatase 1 ◽  
Calyculin A ◽  
Colony Formation Assay ◽  
Effect Of Inhibitors ◽  
Target Enzyme

To investigate the possible involvement of protein phosphatase (PP)1 and PP2A in the process of erythropoiesis, we assessed the effect of PP1 and PP2A inhibitors on erythroid colony formation using an in vitro colony formation assay. Okadaic acid (OKA), calyculin A (Cal-A) and tautomycin suppressed colony formation but 1-nor-okadaone did not. These results suggest that PP1 and PP2A both play an important role in erythropoiesis. Furthermore, higher concentrations of tautomycin were needed to suppress colony formation compared to concentrations of OKA and Cal-A. The target enzyme of inhibitors in erythropoiesis may be PP2A.

Evidence that the endogenous histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not protein phosphatase 2A

1996 ◽  
Vol 109 (6) ◽  
pp. 1437-1447
Author(s):  
J.R. Paulson ◽  
J.S. Patzlaff ◽  
A.J. Vallis
Keyword(s):  
Okadaic Acid ◽  
Hela Cells ◽  
Protein Phosphatase ◽  
Histone H1 ◽  
Protein Kinase Activity ◽  
Mitotic Chromosomes ◽  
Acid Sensitivity ◽  
Unknown Protein

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.

scholarly journals Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein

1995 ◽  
Vol 306 (3) ◽  
pp. 657-665 ◽  
Author(s):  
A Takai ◽  
K Sasaki ◽  
H Nagai ◽  
G Mieskes ◽  
M Isobe ◽  
...  
Keyword(s):  
Dissociation Constant ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Specific Binding ◽  
Protein Phosphatase 2A ◽  
Tight Binding ◽  
Calyculin A ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Phosphatase 2A

Several groups have reported that okadaic acid (OA) and some other tight-binding protein phosphatase inhibitors including microcystin-LR (MCLR), calyculin-A and tautomycin prevent each other from binding to protein phosphatase 2A (PP2A). In this paper, we have introduced an improved procedure for examining to what extent the affinity of an enzyme for a labelled tight-binding ligand is reduced by binding of an unlabelled tight-binding, ligand to the enzyme. Using this procedure, we have analysed the dose-dependent reduction of PP2A binding of [24-3H]OA by addition of OA, MCLR, calyculin-A and tautomycin. The results indicate that the binding of the unlabelled inhibitors to the PP2A molecule causes a dramatic (10(6)-10(8)-fold) increase in the dissociation constant associated with the interaction of [24-3H]OA and PP2A. This suggests that OA and the other inhibitors bind to PP2A in a mutually exclusive manner. The protein phosphatase inhibitors may share the same binding site on the PP2A molecule. We have also measured values of the dissociation constant (Ki) for the interaction of these toxins with protein phosphatase 1 (PP1). For MCLR and calyculin-A, the ratio of the Ki value obtained for PP1 to that for PP2A was in the range 4-9, whereas it was 0.01-0.02 for tautomycin. The value of tautomycin is considerably smaller than that (0.4) calculated from previously reported Ki values.

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