A Novel Phosphoprotein Inhibitor of Protein Type-1 Phosphatase Holoenzymes†

Biochemistry ◽  
1999 ◽  
Vol 38 (51) ◽  
pp. 16952-16957 ◽  
Author(s):  
Masumi Eto ◽  
Andrei Karginov ◽  
David L. Brautigan
Keyword(s):  
Type 1 Phosphatase

Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas

1996 ◽  
Vol 109 (7) ◽  
pp. 1899-1907 ◽  
Author(s):  
G. Habermacher ◽  
W.S. Sale
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Double Mutant ◽  
Sliding Velocity ◽  
Wild Type ◽  
Phosphatase Inhibitors ◽  
Radial Spokes ◽  
Gamma S ◽  
Type 1 Phosphatase

Physiological studies have demonstrated that flagellar radial spokes regulate inner arm dynein activity in Chlamydomonas and that an axonemal cAMP-dependent kinase inhibits dynein activity in radial spoke defective axonemes. These studies also suggested that an axonemal protein phosphatase is required for activation of flagellar dynein. We tested whether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking radial spokes. As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATP gamma S maintained the slow dynein-driven microtubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequent addition of PKI. Preincubation of spoke defective axonemes with the phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 also potently blocked PKI-induced activation of microtubule sliding velocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not. ATP gamma S or the phosphatase inhibitors blocked activation of dynein in a double mutant lacking the radial spokes and the outer dynein arms (pf14pf28). We concluded that the axoneme contains a type-1 phosphatase required for activation of inner arm dynein. We postulated that the radial spokes regulate dynein through the activity of the type-1 protein phosphatase. To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28). As described previously, microtubule sliding velocity was increased from approximately 2 microns/second to approximately 7 microns/second when inner arm dynein from pf14pf28 axonemes ws reconstituted with axonemes containing wild-type spokes. In contrast, pretreatment of inner arm dynein from pf14pf28 axonemes with ATP gamma S, or reconstitution in the presence of microcystin-LR, blocked increased velocity following reconstitution, despite the presence of wild-type radial spokes. We conclude that the radial spokes, through the activity of an axonemal type-1 phosphatase, activate inner arm dynein by dephosphorylation of a critical dynein component. Wild-type radial spokes also operate to inhibit the axonemal cAMP-dependent kinase, which would otherwise inhibit axonemal dynein and motility.

scholarly journals Stimulation of membrane serine-threonine phosphatase in erythrocytes by hydrogen peroxide and staurosporine

1998 ◽  
Vol 274 (2) ◽  
pp. C440-C446 ◽  
Author(s):  
Isabel Bize ◽  
Patricia Muñoz ◽  
Mitzy Canessa ◽  
Philip B. Dunham
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Glycogen Phosphorylase ◽  
Kinase Inhibitor ◽  
Indirect Evidence ◽  
Phosphatase Inhibitors ◽  
Low Sensitivity ◽  
Type 1 Phosphatase ◽  
Stimulation Of

Indirect evidence has suggested that K-Cl cotransport in human and sheep erythrocytes is activated physiologically by a serine-threonine phosphatase. It is activated experimentally by H2O2and by staurosporine, a kinase inhibitor. Activation by H2O2and staurosporine is inhibited by serine-threonine phosphatase inhibitors, suggesting that the activators stimulate the phosphatase. The present study shows that sheep and human erythrocytes contain membrane-associated as well as cytosolic serine-threonine phosphatases, assayed from the dephosphorylation of32P-labeled glycogen phosphorylase. In cells from both species, the relatively low sensitivity of the membrane enzyme to okadaic acid suggests it is type 1 protein phosphatase. The cytosolic phosphatase was much more sensitive to okadaic acid. Membrane-associated phosphatase was stimulated by both H2O2and staurosporine. The results support earlier conclusions that the membrane-associated type 1 phosphatase identified here is regulated by phosphorylation and oxidation. The results are consistent with the phosphatase, or a portion of it, being responsible for activating K-Cl cotransport.

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.

scholarly journals Protein phosphatase composition in the smooth muscle of guinea-pig ileum studied with okadaic acid and inhibitor 2

1989 ◽  
Vol 262 (2) ◽  
pp. 617-623 ◽  
Author(s):  
A Takai ◽  
M Troschka ◽  
G Mieskes ◽  
A V Somlyo
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Okadaic Acid ◽  
Total Activity ◽  
Guinea Pig Ileum ◽  
Control Activity ◽  
Triton X ◽  
Type 1 Phosphatase ◽  
Intracellular Structure

Using okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases, and inhibitor 2, an intrinsic inhibitory factor of type 1 phosphatase, we characterized the phosphorylated myosin light-chain (PMLC) phosphatase activity in the smooth-muscle extracts of guinea-pig ileum. In the intact fibres the control activity was 254 +/- 13 nmol of Pi/min per g wet wt. (n = 15) against 32P-labelled PMLC (4 microM) from chicken gizzard. The following phosphatase fractions were identified: an inhibitor-2-sensitive (type 1) fraction (fractional activity = 35%), a Mg2+-dependent and okadaic acid-insensitive (type 2C) fraction (17%), and two type 2A-like fractions that had different susceptibility to okadaic acid. The type 2A-like fraction with lower affinity to okadaic acid accounted for 30% of the control activity. After the cell membrane was permeabilized by Triton X-100, more than 60% of this fraction remained and accounted for about 90% of the total activity, whereas the other fractions were nearly abolished. The type 2A-like fraction may be bound to some intracellular structure such as contractile proteins.

scholarly journals Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.

1990 ◽  
Vol 111 (1) ◽  
pp. 103-112 ◽  
Author(s):  
A Fernandez ◽  
D L Brautigan ◽  
M Mumby ◽  
N J Lamb
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Metabolic Labeling ◽  
Actin Microfilaments ◽  
Actin Microfilament ◽  
Nonmuscle Cells ◽  
Dependent Protein ◽  
Type 1 Phosphatase

Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.

scholarly journals Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p

1999 ◽  
Vol 13 (5) ◽  
pp. 545-555 ◽  
Author(s):  
I. Sassoon ◽  
F. F. Severin ◽  
P. D. Andrews ◽  
M.-R. Taba ◽  
K. B. Kaplan ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type 1 Phosphatase

Role of Ca2+/calmodulin-dependent phosphatase 2B in thrombin-induced endothelial cell contractile responses

1998 ◽  
Vol 275 (4) ◽  
pp. L788-L799 ◽  
Author(s):  
Alexander D. Verin ◽  
Clare Cooke ◽  
Maria Herenyiova ◽  
Carolyn E. Patterson ◽  
Joe G. N. Garcia
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Cytoskeletal Protein ◽  
Barrier Dysfunction ◽  
Western Immunoblotting ◽  
Protein Dephosphorylation ◽  
Type 1 Phosphatase

Thrombin-induced Ca2+mobilization, activation of Ca2+/calmodulin-dependent myosin light chain (MLC) kinase (MLCK), and increased phosphorylation of MLCs precede and are critical to endothelial cell (EC) barrier dysfunction. Net MLC dephosphorylation after thrombin is nearly complete by 60 min and involves type 1 phosphatase (PPase 1) activity. We now report that thrombin does not alter total PPase 1 activity in EC homogenates but rather decreases myosin-associated PPase 1 activity. The PPase 1 inhibitor calyculin fails to prevent thrombin-induced MLC dephosphorylation. However, thrombin significantly increased the activity of Ca2+-dependent PPase 2B in EC homogenates (∼1.5- to 2-fold), with PPase 2B activation correlating with phosphorylation of the PPase 2B catalytic subunit. Western immunoblotting revealed PPase 2B to be present in cytoskeletal EC fractions, with specific PPase 2B inhibitors such as cyclosporin (200 nM) and deltamethrin (100 nM to 1 μM) attenuating thrombin-induced cytoskeletal protein dephosphorylation, including EC MLC dephosphorylation. These results suggest a model whereby thrombin-inducible contraction is determined by the phosphorylation status of EC MLC regulated by the balance between EC MLCK, PPase 1 (constitutive), and PPase 2B (inducible) activities.

scholarly journals YPI1 and SDS22 Proteins Regulate the Nuclear Localization and Function of Yeast Type 1 Phosphatase Glc7

2006 ◽  
Vol 282 (5) ◽  
pp. 3282-3292 ◽  
Author(s):  
Leda Pedelini ◽  
Maribel Marquina ◽  
Joaquin Ariño ◽  
Antonio Casamayor ◽  
Libia Sanz ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
And Function ◽  
Type 1 Phosphatase
Sign in / Sign up

Export Citation Format

Share Document