scholarly journals Reconstruction of spatial structure of plant protein phosphatase type 1, 2a and 4 in complexes with microcystin-LR

1970 ◽  
pp. 339-344
Author(s):  
D. A. Samofalova ◽  
P. A. Karpov ◽  
O. V. Raievskyi ◽  
Ya. B. Blume
Keyword(s):  
Molecular Docking ◽  
Spatial Structure ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Specific Interaction ◽  
Specific Protein ◽  
Plant Protein ◽  
Scoring Functions ◽  
High Level

Aim. The major toxicity of Microcystin-LR (MCLR) has been ascribed to its potent ability to inhibit serine/threonine-specific protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). Although MCLR is widely used in animal models its selectivity for these enzymes of plant origin is not still investigated in details for phylogenetically diversified sources. Methods. The spatial structure of plant PP1, PP2A, PP4 protein phosphatases was reconstructed with homology modeling method. Flexible docking of MCLR was performed using CCDC GOLD Suite 5.3. For docking evaluations, GOLD scoring functions were used. Results. Information about amino acids, involved in ligand binding, was obtained from 8 experimentally proved human MCLR-PP1 and PP2A complexes. The sites of microcystin-LR binding with plant protein phosphatases (type-1, 2A and 4) were proved by comparative analysis and molecular docking. A high level of sequence and structure identity of plant and animal phosphatases allow us to conclude similarity of MCLR binding in PP1, PP2A and PP4. Keywords: microcystin-LR, protein phosphatase, specific interaction, molecular docking.

scholarly journals Role for protein phosphatases in the cell-cycle-regulated phosphorylation of stathmin

1998 ◽  
Vol 334 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Sucharita J. MISTRY ◽  
Heng-Chun LI ◽  
George F. ATWEH
Keyword(s):  
Cell Cycle ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Specific Protein ◽  
G1 Phase ◽  
Major Increase ◽  
Protein Phosphatase Type

Stathmin is a major cytosolic phosphoprotein that regulates microtubule dynamics during the assembly of the mitotic spindle. The activity of stathmin itself is regulated by changes in its state of phosphorylation during the transition from interphase to metaphase. For a better understanding of the regulation of stathmin activity during the cell cycle, we explored the mechanism(s) responsible for the decrease in the level of phosphorylation of stathmin as cells complete mitosis and enter a new G1 phase. We show that stathmin mRNA and protein are expressed constitutively throughout the different phases of the cell cycle. This suggests that the non-phosphorylated stathmin that predominates during G1 is not generated by degradation of phosphorylated stathmin in mitosis and synthesis of new unphosphorylated stathmin as cells enter a new G1 phase. This suggested that protein phosphatases might be responsible for dephosphorylating stathmin as cells enter a new cell cycle. Okadaic acid-mediated inhibition of protein phosphatases in vivoshowed a major increase in the level of phosphorylation of stathmin. Dephosphorylation studies in vitro showed differential patterns of site-specific dephosphorylaton of stathmin to protein phosphatase type 1, protein phosphatase type 2A and protein phosphatase type 2B. Thus stathmin might be a target for okadaic acid-sensitive protein phosphatase(s), and its activity in eukaryotic cells might be modulated by the sequential activity of specific protein kinases and phosphatases.

Effects of Serine/Threonine Protein Phosphatases on Ion Channels in Excitable Membranes

2000 ◽  
Vol 80 (1) ◽  
pp. 173-210 ◽  
Author(s):  
Stefan Herzig ◽  
Joachim Neumann
Keyword(s):  
Ion Channels ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Direct Interaction ◽  
Specific Protein ◽  
Biochemical Properties ◽  
Future Research ◽  
Regulatory Subunits ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors

This review deals with the influence of serine/threonine-specific protein phosphatases on the function of ion channels in the plasma membrane of excitable tissues. Particular focus is given to developments of the past decade. Most of the electrophysiological experiments have been performed with protein phosphatase inhibitors. Therefore, a synopsis is required incorporating issues from biochemistry, pharmacology, and electrophysiology. First, we summarize the structural and biochemical properties of protein phosphatase (types 1, 2A, 2B, 2C, and 3–7) catalytic subunits and their regulatory subunits. Then the available pharmacological tools (protein inhibitors, nonprotein inhibitors, and activators) are introduced. The use of these inhibitors is discussed based on their biochemical selectivity and a number of methodological caveats. The next section reviews the effects of these tools on various classes of ion channels (i.e., voltage-gated Ca2+ and Na+ channels, various K+ channels, ligand-gated channels, and anion channels). We delineate in which cases a direct interaction between a protein phosphatase and a given channel has been proven and where a more complex regulation is likely involved. Finally, we present ideas for future research and possible pathophysiological implications.

scholarly journals Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases

1991 ◽  
Vol 275 (1) ◽  
pp. 233-239 ◽  
Author(s):  
A Takai ◽  
G Mieskes
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Inhibitory Effect ◽  
Enzyme Concentration ◽  
Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Type 1 Phosphatase

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.

Reconstitution of calyculin-inhibited K-Cl cotransport in dog erythrocyte ghosts by exogenous PP-1

1996 ◽  
Vol 270 (3) ◽  
pp. C898-C902 ◽  
Author(s):  
T. Krarup ◽  
P. B. Dunham
Keyword(s):  
Protein Phosphatase ◽  
Direct Evidence ◽  
Potent Inhibitor ◽  
Protein Phosphatases ◽  
Calyculin A ◽  
Osmotic Swelling ◽  
Maximal Activation ◽  
Resealed Ghosts ◽  
K Transport

Osmotic swelling of dog and other mammalian erythrocytes activates Cl-dependent K transport, K-Cl cotransport. This activation can be abolished by nanomolar concentrations of calyculin, a potent inhibitor of serine-threonine protein phosphatases. Therefore, K-Cl cotransport is probably activated by dephosphorylation by a type 1 and/or type 2A protein phosphatase (PP-1 and PP-2A, respectively). This was tested directly by incorporating exogenous protein phosphatases into resealed ghosts made from dog erythrocytes previously exposed to calyculin. K-Cl cotransport was nearly completely inhibited in the ghosts. Incorporation of PP-1 reconstituted K-Cl cotransport. Maximal reconstitution was up to 90% of the control flux in the ghosts and 0.1 U PP-1/ml lysate gave half-maximal reconstitution of cotransport. In contrast, PP-2A had no effect. This result with PP-1 provides direct evidence that K-Cl cotransport is activated by PP-1 in dog erythrocytes. Half-maximal activation of K-Cl cotransport required approximately 180 molecules of PP-1 per ghost.

scholarly journals Discovery of a protein phosphatase activity encoded in the genome of bacteriophage λ. Probable identity with open reading frame 221

1989 ◽  
Vol 260 (3) ◽  
pp. 931-934 ◽  
Author(s):  
P T W Cohen ◽  
P Cohen
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Glycogen Synthase ◽  
Sequence Similarity ◽  
Protein Phosphatases ◽  
Specific Protein ◽  
Open Reading Frame ◽  
Phage Lambda ◽  
Reading Frame

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.

Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes

1999 ◽  
Vol 277 (5) ◽  
pp. C926-C936 ◽  
Author(s):  
Isabel Bize ◽  
Birol Güvenç ◽  
Aeisha Robb ◽  
Guido Buchbinder ◽  
Carlo Brugnara
Keyword(s):  
Ionic Strength ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Human Erythrocytes ◽  
Phosphatase Inhibitors ◽  
Pp2a Activity ◽  
Highly Sensitive ◽  
Protein Phosphatase Inhibitors ◽  
Membrane Bound

Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 ± 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 ± 10% per 100 meq/l). Increases in membrane PP2A activity (270 ± 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 ± 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes.

Molecular mechanisms underlying the interaction of motuporin and microcystins with type-1 and type-2A protein phosphatases

1996 ◽  
Vol 74 (4) ◽  
pp. 569-578 ◽  
Author(s):  
Marcia Craig ◽  
Hue Anh Luu ◽  
Tara L. McCready ◽  
Charles F. B. Holmes ◽  
David Williams ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Molecular Mechanisms ◽  
Protein Phosphatases ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Striking Difference ◽  
Functional Difference ◽  
Subsequent Formation ◽  
Covalent Adduct

Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA, and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase–toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and noduIarin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.Key words: microcystins, nodularin, motuporin, protein phosphatases, protein phosphorylation.

Okadaic acid inhibits activation of K-Cl cotransport in red blood cells containing hemoglobins S and C

1991 ◽  
Vol 261 (4) ◽  
pp. C591-C593 ◽  
Author(s):  
E. P. Orringer ◽  
J. S. Brockenbrough ◽  
J. A. Whitney ◽  
P. S. Glosson ◽  
J. C. Parker
Keyword(s):  
Red Blood Cells ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Blood Cells ◽  
Specific Protein ◽  
Cell Swelling ◽  
Osmotic Swelling ◽  
New Information ◽  
Normal Humans

The sensitivity of red blood cells containing hemoglobins S and C to activation of K-Cl cotransport by osmotic swelling and acidification was reduced by okadaic acid, a specific protein phosphatase inhibitor. The dose-response curve for okadaic acid suggests its action is on a type 1 protein phosphatase. Okadaic acid has been previously shown to inhibit swelling-induced activation of K-Cl cotransport in red blood cells from rabbits, normal humans, and dogs. The present work confirms the observation that okadaic acid blunts the stimulation of K-Cl cotransport by cell swelling. The new information is that okadaic acid reduces the effects of hemoglobins S and C on the volume and pH sensitivity of K-Cl cotransport. Thus the influences of cell volume, pH, and mutant hemoglobins may all be mediated via a common mechanisms that affects the phosphorylation state, either of the K-Cl. cotransporter itself or of a protein that regulates its function.

scholarly journals Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission

2019 ◽  
Vol 15 (7) ◽  
pp. e1007973 ◽  
Author(s):  
Thomas Hollin ◽  
Caroline De Witte ◽  
Aline Fréville ◽  
Ida Chiara Guerrera ◽  
Cerina Chhuon ◽  
...  
Keyword(s):  
Plasmodium Berghei ◽  
Protein Phosphatase ◽  
Essential Role ◽  
Specific Protein ◽  
Protein Phosphatase Type 1 ◽  
Protein Phosphatase Type

Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

Diabetes ◽  
1994 ◽  
Vol 43 (10) ◽  
pp. 1234-1241 ◽  
Author(s):  
Y. H. Chen ◽  
L. Hansen ◽  
M. X. Chen ◽  
C. Bjorbaek ◽  
H. Vestergaard ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mrna Level ◽  
Regulatory Subunit ◽  
Coding Region
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