scholarly journals Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide.

1991 ◽  
Vol 97 (4) ◽  
pp. 799-817 ◽  
Author(s):  
M L Jennings ◽  
R K Schulz
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Direct Action ◽  
Transport Protein ◽  
Cell Swelling ◽  
Kcl Cotransport ◽  
Rapid Removal ◽  
Protein Dephosphorylation ◽  
Protein Kinase Kinase

The mechanism of activation of KCl cotransport has been examined in rabbit red blood cells. Previous work has provided evidence that a net dephosphorylation is required for activation of transport by cell swelling. In the present study okadaic acid, an inhibitor of protein phosphatases, was used to test this idea in more detail. We find that okadaic acid strongly inhibits swelling-stimulated KCl cotransport. The IC50 for okadaic acid is approximately 40 nM, consistent with the involvement of type 1 protein phosphatase in transport activation. N-Ethylmaleimide (NEM) is well known to activate KCl cotransport in cells of normal volume. Okadaic acid, added before NEM, inhibits the activation of transport by NEM, indicating that a dephosphorylation is necessary for the NEM effect. Okadaic acid added after NEM inhibits transport only very slightly. After a brief exposure to NEM and rapid removal of unreacted NEM, KCl cotransport activates with a time delay that is similar to that for swelling activation. Okadaic acid causes a slight increase in the delay time. These findings are all consistent with the idea that NEM activates transport not by a direct action on the transport protein but by altering a phosphorylation-dephosphorylation cycle. The simplest hypothesis that is consistent with the data is that both cell swelling and NEM cause inhibition of a protein kinase. Kinase inhibition causes net dephosphorylation of some key substrate (not necessarily the transport protein); dephosphorylation of this substrate, probably by type 1 protein phosphatase, causes transport activation.

K-Cl cotransport in rabbit red cells: further evidence for regulation by protein phosphatase type 1

1993 ◽  
Vol 264 (1) ◽  
pp. C118-C124 ◽  
Author(s):  
L. C. Starke ◽  
M. L. Jennings
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Inhibitory Concentration ◽  
Cell Swelling ◽  
Calyculin A ◽  
Phosphatase Inhibitor ◽  
Protein Phosphatase Type 1 ◽  
Pharmacological Studies ◽  
Protein Phosphatase Type

We have examined inhibition of swelling-induced K-Cl cotransport in rabbit red blood cells by calyculin A, a potent serine-threonine protein phosphatase inhibitor, to determine whether transport is regulated by phosphatase type 1 or type 2A. Calyculin A blocks K(Rb) influx [half-maximal inhibitory concentration (IC50) = 3-6 nM] 10 times more potently than a second phosphatase inhibitor, okadaic acid (IC50 = 40 nM), consistent with earlier pharmacological studies showing that calyculin A inhibits phosphatase type 1 10 times more effectively than does okadaic acid. Calyculin A always inhibits Rb influx when added either before or after cell swelling, indicating that the phosphatase must operate continually to first activate and then maintain high transport rates in swollen cells. Similarly, N-ethylmaleimide (NEM) fails to stimulate K-Cl cotransport only when added to cells pretreated with calyculin A. Therefore, like cell swelling, activation of K-Cl cotransport by NEM involves a phosphatase sensitive to calyculin A. We conclude that cell swelling and NEM activate K-Cl cotransport via a net dephosphorylation that appears to involve protein phosphatase type 1.

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.

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 Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

Reproduction ◽  
2006 ◽  
Vol 131 (6) ◽  
pp. 1017-1024 ◽  
Author(s):  
K Ashizawa ◽  
G J Wishart ◽  
S Katayama ◽  
D Takano ◽  
A R A H Ranasinghe ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Acrosome Reaction ◽  
Dependent Manner ◽  
Kinase C ◽  
Protein Phosphatase Type 1 ◽  
Protein Phosphatase Type

The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 °C. In the presence of 2 mmol CaCl2/l at 40 °C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca2+, motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 °C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10–1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca2+. These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca2+, was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca2+ and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca2+ plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.

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.

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
Sign in / Sign up

Export Citation Format

Share Document