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.

scholarly journals Blocking Protein Phosphatase 1 [PP1] Prevents Loss of Tether Elasticity in Anaphase Crane-Fly Spermatocytes

2021 ◽  
Vol 8 ◽  
Author(s):  
Arthur Forer ◽  
Aisha Adil ◽  
Michael W. Berns
Keyword(s):  
Protein Phosphatase ◽  
Chromosome Pair ◽  
Protein Phosphatases ◽  
Protein Phosphatase 1 ◽  
Calyculin A ◽  
Early Anaphase ◽  
In Cells

In normal anaphase cells, telomeres of each separating chromosome pair are connected to each other by tethers. Tethers are elastic at the start of anaphase: arm fragments cut from anaphase chromosomes in early anaphase move across the equator to the oppositely-moving chromosome, telomere moving toward telomere. Tethers become inelastic later in anaphase as the tethers become longer: arm fragments no longer move to their partners. When early anaphase cells are treated with Calyculin A (CalA), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), at the end of anaphase chromosomes move backward from the poles, with telomeres moving toward partner telomeres. Experiments described herein show that in cells treated with CalA, backwards movements are stopped in a variety of ways, by cutting the tethers of backwards moving chromosomes, by severing arms of backwards moving chromosomes, by severing arms before the chromosomes reach the poles, and by cutting the telomere toward which a chromosome is moving backwards. Measurements of arm-fragment velocities show that CalA prevents tethers from becoming inelastic as they lengthen. Since treatment with CalA causes tethers to remain elastic throughout anaphase and since inhibitors of PP2A do not cause the backwards movements, PP1 activity during anaphase causes the tethers to become inelastic.

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.

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.

Ca2+ and calmodulin-dependent protein phosphatase from Leishmania donovani

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 567-573 ◽  
Author(s):  
C. BANERJEE ◽  
D. SARKAR ◽  
A. BHADURI
Keyword(s):  
Cyclosporin A ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Leishmania Donovani ◽  
Protein Phosphatases ◽  
Cross Reactivity ◽  
Calmodulin Antagonists ◽  
Type Protein ◽  
Complete Obliteration ◽  
Dependent Protein

A protein phosphatase exclusively dependent upon micromolar amounts of Ca2+ and calmodulin has been identified and partially purified from Leishmania spp. Complete obliteration of its activity is observed in the presence of calmodulin antagonists such as trifluoperazine, fluphenazine and calmidazolium. Relative insensitivity to okadaic acid and lack of activation in the absence of Ca2+ and calmodulin distinguishes this enzyme from PP1, PP2A and PP2C-type protein phosphatases. Cross-reactivity of the enzyme was observed with antibodies that recognize both the A and B chains of calcineurin, a PP2B type Ca2+ and calmodulin-dependent phosphatase from brain. FK506, an immunosuppresive drug that inhibits the enzyme from other sources inhibited the enzyme only in the presence of exogenous FK binding protein, whereas Cyclosporin A inhibited the enzyme in crude preparations. Taken together these results reveal the presence of a Ca2+ and calmodulin-dependent phosphatase from Leishmania. This is the first report of the presence of a PP2B-type protein phosphatase from a pathogenic protozoa.

Inhibitory effect of calyculin A, a Ser/Thr protein phosphatase type I inhibitor, on renin secretion

1998 ◽  
Vol 275 (5) ◽  
pp. F664-F670 ◽  
Author(s):  
Chun Sik Park ◽  
Mi Hyun Kim ◽  
Chae Hun Leem ◽  
Yeon Jin Jang ◽  
Hae Won Kim ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Renin Secretion ◽  
Type I ◽  
Dependent Manner ◽  
Calyculin A ◽  
Selective Inhibitors ◽  
Intracellular Ca

We have recently shown that several putative selective inhibitors of Ca2+-calmodulin-dependent myosin light chain kinase (MLCK), such as ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], reversibly stimulate renin secretion [C. S. Park, S.-H. Chang, H. S. Lee, S.-H. Kim, J. W. Chang, and C. D. Hong. Am. J. Physiol. 271 ( Cell Physiol. 40): C242–C247, 1996]. We hypothesized that Ca2+ inhibits renin secretion, via phosphorylation of 20-kDa myosin light chain (MLC20), by activating MLCK. In the present studies, we have investigated the types of protein phosphatase (PP) involved in the control of renin secretion through inhibition of MLC dephosphorylation using inhibitors of various types of serine/threonine-specific protein phosphatases. Cyclosporin A, a putative inhibitor of PP type 2 (calcineurin), was without effect. Calyculin A and okadaic acid, putative selective inhibitors of both PP type 1 (PP1) and type 2A (PP2A), significantly inhibited renin secretion under control conditions. Calyculin A had inhibitory effects at least 10-fold more potent than okadaic acid, suggesting that PP1, rather than PP2A, is involved in the control of renin secretion. Furthermore, calyculin A blocked the reversal of renin secretion preinhibited by raised intracellular Ca2+ concentrations in a concentration-dependent manner. Calyculin A (10−6 M) significantly inhibited renin secretion stimulated by lowering intracellular Ca2+ concentrations and blocked the stimulatory effect of ML-9 on renin secretion. Taking all of these results into consideration, we hypothesize that dephosphorylation of MLC20 by Ca2+-independent PP1 stimulates renin secretion, whereas phosphorylation of MLC20 by Ca2+-calmodulin-dependent MLCK inhibits it. This hypothesized regulatory model of renin secretion predicts that the rate of renin secretion at a given time is determined by the ratio of phosphorylated to dephosphorylated MLC20, which is, in turn, determined by the dynamic balance between activity of MLCK and MLC phosphatase.

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