scholarly journals Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A

1999 ◽  
Vol 341 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Svante RESJÖ ◽  
Alina OKNIANSKA ◽  
Stanislaw ZOLNIEROWICZ ◽  
Vincent MANGANIELLO ◽  
Eva DEGERMAN
Keyword(s):  
Cyclosporin A ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Calyculin A ◽  
Phosphatase Inhibitors ◽  
Pp2a Activity ◽  
Phosphodiesterase Type ◽  
Fold Activation ◽  
Protein Phosphatase Type

Phosphodiesterase type 3B (PDE3B) has been shown to be activated and phosphorylated in response to insulin and hormones that increase cAMP. In order to study serine/threonine protein phosphatases involved in the regulation of rat adipocyte PDE3B, we investigated the phosphorylation and activation of PDE3B in vivoin response to phosphatase inhibitors and the dephosphorylation and deactivation of PDE3B in vitroby phosphatases purified from rat adipocyte homogenates. Okadaic acid and calyculin A induced dose- and time-dependent activation of PDE3B. Maximal effects were obtained after 30 min using 1 μM okadaic acid (1.8-fold activation) and 300 nM calyculin A (4-fold activation), respectively. Tautomycin and cyclosporin A did not induce activation of PDE3B. Incubation of adipocytes with 300 nM calyculin A inhibited protein phosphatase (PP) 1 and PP2A completely. Okadaic acid (1 μM) reduced PP2A activity by approx. 50% but did not affect PP1 activity, and 1 μM tautomycin reduced PP1 activity by approx. 60% but PP2A activity by only 11%. This indicates an important role for PP2A in the regulation of PDE3B. Furthermore, rat adipocyte PDE3B phosphatase activity co-purified with PP2A but not with PP1 during MonoQ chromatography. As compared with insulin, okadaic acid and calyculin A induced phosphorylation of PDE3B by 2.8- and 14-fold respectively, whereas tautomycin and cyclosporin A had no effect. Both calyculin A and okadaic acid induced phosphorylation on serine 302, the site known to be phosphorylated on PDE3B in response to insulin and isoproterenol (isoprenaline), as well as on sites not identified previously. In summary, PP2A seems to be involved in the regulation of PDE3B in vivoand can act as a PDE3B phosphatase in vitro. In comparison with insulin, calyculin A induced a dramatic activation of PDE3B and both calyculin A and okadaic acid induced phosphorylation on additional sites, which could have a role in signalling pathways not yet identified.

Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1081-1087 ◽  
Author(s):  
Robert A. R. Hurta ◽  
Jim A. Wright
Keyword(s):  
Okadaic Acid ◽  
Ribonucleotide Reductase ◽  
Protein Phosphatase ◽  
Reductase Activity ◽  
Tumor Promoter ◽  
Calyculin A ◽  
Tumor Promoters ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Ribonucleotide Reductase Activity

A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment. This first demonstration that the non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause rapid alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, particularly the R2 component, plays a fundamental role in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis.Key words: ribonucleotide reductase, DNA synthesis, okadaic acid, calyculin A, tumor promoter, protein phosphatase.

scholarly journals Effects of the Protein Phosphatase Inhibitors Okadaic Acid and Calyculin A on Insulin Release from Rat Pancreatic Islets.

1992 ◽  
Vol 39 (3) ◽  
pp. 325-329 ◽  
Author(s):  
TATSUO TAMAGAWA ◽  
AKIHISA IGUCHI ◽  
KAZUMASA UEMURA ◽  
HISAYUKI MIURA ◽  
KATSUNORI NONOGAKI ◽  
...  
Keyword(s):  
Pancreatic Islets ◽  
Insulin Release ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Calyculin A ◽  
Phosphatase Inhibitors ◽  
Rat Pancreatic Islets ◽  
Protein Phosphatase Inhibitors

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.

scholarly journals Colorimetric Immuno-Protein Phosphatase Inhibition Assay for Specific Detection of Microcystins and Nodularins of Cyanobacteria

2001 ◽  
Vol 67 (2) ◽  
pp. 904-909 ◽  
Author(s):  
James S. Metcalf ◽  
Steven G. Bell ◽  
Geoffrey A. Codd
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cyclic Peptide ◽  
Calyculin A ◽  
Inhibition Assay ◽  
Specific Detection ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibition ◽  
Phosphatase Inhibition

ABSTRACT A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp3-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R 2 = 0.94, P< 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins.

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.

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