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.

scholarly journals Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts.

1993 ◽  
Vol 4 (4) ◽  
pp. 397-411 ◽  
Author(s):  
P R Clarke ◽  
I Hoffmann ◽  
G Draetta ◽  
E Karsenti
Keyword(s):  
Protein Kinase ◽  
Protein Phosphatase ◽  
Cyclin B ◽  
Specific Inhibition ◽  
Kinase Activation ◽  
Protein Kinase Activation ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Low Activity

We have examined the roles of type-1 (PP-1) and type-2A (PP-2A) protein-serine/threonine phosphatases in the mechanism of activation of p34cdc2/cyclin B protein kinase in Xenopus egg extracts. p34cdc2/cyclin B is prematurely activated in the extracts by inhibition of PP-2A by okadaic acid but not by specific inhibition of PP-1 by inhibitor-2. Activation of the kinase can be blocked by addition of the purified catalytic subunit of PP-2A at a twofold excess over the activity in the extract. The catalytic subunit of PP-1 can also block kinase activation, but very high levels of activity are required. Activation of p34cdc2/cyclin B protein kinase requires dephosphorylation of p34cdc2 on Tyr15. This reaction is catalysed by cdc25-C phosphatase that is itself activated by phosphorylation. We show that, in interphase extracts, inhibition of PP-2A by okadaic acid completely blocks cdc25-C dephosphorylation, whereas inhibition of PP-1 by specific inhibitors has no effect. This indicates that a type-2A protein phosphatase negatively regulates p34cdc2/cyclin B protein kinase activation primarily by maintaining cdc25-C phosphatase in a dephosphorylated, low activity state. In extracts containing active p34cdc2/cyclin B protein kinase, dephosphorylation of cdc25-C is inhibited, whereas the activity of PP-2A (and PP-1) towards other substrates is unaffected. We propose that this specific inhibition of cdc25-C dephosphorylation is part of a positive feedback loop that also involves direct phosphorylation and activation of cdc25-C by p34cdc2/cyclin B. Dephosphorylation of cdc25-C is also inhibited when cyclin A-dependent protein kinase is active, and this may explain the potentiation of p34cdc2/cyclin B protein kinase activation by cyclin A. In extracts supplemented with nuclei, the block on p34cdc2/cyclin B activation by unreplicated DNA is abolished when PP-2A is inhibited or when stably phosphorylated cdc25-C is added, but not when PP-1 is specifically inhibited. This suggests that unreplicated DNA inhibits p34cdc2/cyclin B activation by maintaining cdc25-C in a low activity, dephosphorylated state, probably by keeping the activity of a type-2A protein phosphatase towards cdc25-C at a high level.

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.

Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

2007 ◽  
Vol 313 (9) ◽  
pp. 1897-1910 ◽  
Author(s):  
Hiromi Ito ◽  
Yuhei Koyama ◽  
Makoto Takano ◽  
Kohei Ishii ◽  
Mitsugu Maeno ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Protein phosphatase 1 regulates the cytoplasmic dynein-driven formation of endoplasmic reticulum networks in vitro.

1995 ◽  
Vol 128 (5) ◽  
pp. 879-891 ◽  
Author(s):  
V Allan
Keyword(s):  
Endoplasmic Reticulum ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Kinase Inhibitor ◽  
Cytoplasmic Dynein ◽  
Protein Phosphatase 1 ◽  
Egg Extracts ◽  
Vanadate Inhibition ◽  
Membrane Networks

Interphase Xenopus egg extracts form extensive tubular membrane networks in vitro. These networks are identified here as endoplasmic reticulum by the presence of ER resident proteins, as shown by immunofluorescence, and by the presence of single ribosomes and polysomes, as shown by electron microscopy. The effect of phosphorylation on ER movement in interphase was tested using the phosphatase inhibitor, okadaic acid. Okadaic acid treatment resulted in an increase of up to 27-fold in the number of ER tubules moving and in the extent of ER networks formed compared to control extracts. This activation was blocked by the broad-specificity kinase inhibitor 6-dimethylaminopurine. Okadaic acid had no effect, however, on the direction of ER tubule movement, which occurred towards the minus end of microtubules, and was sensitive to low concentrations of vanadate. Inhibition of phosphatases also had no effect on the speed or duration of ER tubule extensions, and did not stimulate the activity of soluble cytoplasmic dynein. The sensitivity of ER movement to okadaic acid closely matched that of protein phosphatase 1. Although the amount of ER motility was greatly increased by inhibiting protein phosphatase 1 (PP1), the amount of cytoplasmic dynein associated with the membrane was not altered. The data support a model in which phosphorylation regulates ER movement by controlling the activity of cytoplasmic dynein bound to the ER membrane.

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.

Stimulatory phosphorylation of cAMP-specific PDE4D5 by contractile agonists is mediated by PKC-dependent inactivation of protein phosphatase 2A

2008 ◽  
Vol 294 (1) ◽  
pp. G327-G335 ◽  
Author(s):  
Karnam S. Murthy ◽  
Wimolpak Sriwai
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Dependent Inactivation ◽  
Rhythmic Cycles ◽  
Phosphatase 2A ◽  
Contraction And Relaxation ◽  
Camp Levels

Smooth muscle of the gut undergoes rhythmic cycles of contraction and relaxation. Various constituents in the pathways that mediate muscle contraction could act to cross-regulate cAMP or cGMP levels and terminate subsequent relaxation. We have previously shown that cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4D5; the latter is the only PDE4D isoform expressed in smooth muscle. In the present study we have elucidated a mechanism whereby cholecystokinin (CCK) and, presumably, other contractile agonists capable of activating PKC can cross-regulate cAMP levels. Forskolin stimulated PDE4D5 phosphorylation and PDE4D5 activity. CCK significantly increased forskolin-stimulated PDE4D5 phosphorylation and activity and attenuated forskolin-stimulated cAMP levels. The effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity and on cAMP levels was blocked by the inhibitors of PLC or PKC and in cultured muscle cells by the expression of Gαq minigene. The effects of CCK on PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were mimicked by low (1 nM) concentrations of okadaic acid, but not by a low (10 nM) concentration of tautomycin, suggesting involvement of PP2A. Purified catalytic subunit of PP2A but not PP1 dephosphorylated PDE4D5 in vitro. Coimmunoprecipitation studies demonstrated association of PDE4D5 with PP2A and the association was decreased by the activation of PKC. In conclusion, cAMP levels are cross-regulated by contractile agonists via a mechanism that involves PLC-β-dependent, PKC-mediated inhibition of PP2A activity that leads to increase in PDE4D5 phosphorylation and activity and inhibition of cAMP levels.

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