Development of an Assay Method for Activities of Serine/Threonine Protein Phosphatase Type 2B (Calcineurin) in Crude Extracts

Mitsuhashi, Shinya; Shima, Hiroshi; Kikuchi, Kunimi; Igarashi, Keiichi; Hatsuse, Rei; Maeda, Kenichi; Yazawa, Michio; Murayama, Toshihiko; Okuma, Yasunobu; Nomura, Yasuyuki

doi:10.1006/abio.1999.4422

Cited by 19 publications

(19 citation statements)

References 37 publications

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“…With a view toward eventual human use in alcoholism treatment this study did not address two further considerations: location of action and side effects. Research in rodents suggests that the CNS striatum, including the caudate nucleus, putamen, nucleus accumbens, and the olfactory tubercle along with the hippocampus (Mitsuhashi, 2000), contain the highest CLN activities in the brain. Whether these sites reflect the locales of CsA and TRL in limiting CLN actions remains to be seen.…”

Section: Discussionmentioning

confidence: 99%

Immunophyllin Ligands Show Differential Effects on Alcohol Self-Administration in C57BL Mice

Beresford

Fay²,

Serkova³

et al. 2012

J Pharmacol Exp Ther

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High abstinence rates characterize alcohol-dependent liver graft recipients. The immunosuppressants cyclosporine A (CsA) and tacrolimus (TRL) also inhibit calcineurin (CLN) in the brain. Previously, we found that CsA reduces alcohol consumption in C57BL/6J mice. The goals of the present study were: 1) to compare the ethanol preference effects of CsA against TRL, as well as sirolimus (SRL), an immunosuppressant without CLN inhibition and 2) to establish that reduction of alcohol consumption is not caused by caloric reinforcement from these ligands. C57BL/6J mice trained to imbibe ethanol consumed ethanol or sucrose in a modified limited-access drinking-in-the-dark paradigm; test groups received vehicle or doses of CsA (5-50 mg/kg), TRL (0.5-2.5 mg/kg), or SRL (1.0 -5.0 mg/kg) for 5 consecutive days, 30 min before each 2-h limited-access session. Brain CsA, TRL, and SRL concentrations were measured. CsA (p Ͻ 0.001) and TRL (p Ͻ 0.01) each decreased ethanol consumption, whereas SRL showed no significant effects at any dose. Effective doses included CsA at 10 mg/kg and above and TRL at 2.5 mg/kg. CsA (50 mg/kg) did not reduce sucrose consumption. Both CsA and TRL reached significant brain concentrations compared with very low values of SRL. These data suggest that CsA and TRL may reduce alcohol preference through central CLN inhibition rather than by immunosuppression.

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Section: Discussionmentioning

confidence: 99%

Immunophyllin Ligands Show Differential Effects on Alcohol Self-Administration in C57BL Mice

Beresford

Fay²,

Serkova³

et al. 2012

J Pharmacol Exp Ther

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“…The supernatant was then placed on a mini Quick Spin Oligo Column (Roche Diagnostics) to remove free phosphates. Calcineurin phosphatase activity was then measured nonradioactively with RII phosphopeptide substrate and Malachite green using a commercial kit and following the manufacturer's instructions (Biomol green cellular calcineurin assay kit plus), except that the lysis buffer and assay buffer were supplemented with 5 mM ascorbic acid (Mitsuhashi et al, 2000).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Human Insulin Gene Transcription by the Immunosuppressive Drugs Cyclosporin A and Tacrolimus in Primary, Mature Islets of Transgenic Mice

et al. 2003

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Cyclosporin A and tacrolimus are clinically important immunosuppressive drugs. They share a diabetogenic action as one of their most serious adverse effects. The underlying mechanism is unknown. Previous studies have shown that tacrolimus can inhibit insulin gene transcription at high concentrations in tumor cell lines. To study insulin gene transcription in normal, mature pancreatic islet cells, we used a novel approach in the present study. Transgenic mice that carry a human insulin promoterreporter gene were generated. The human insulin promoter directed transcription in pancreatic islets and conferred a normal, physiological glucose response to reporter gene expression in isolated islets. After stimulation with glucose, human insulin promoter-mediated gene expression was inhibited in normal, mature islet cells by both tacrolimus and cyclosporin A to a large extent (approximately 70%) and with high potency at concentrations that are known to inhibit calcineurin phosphatase activity (IC 50 values of 1 and 35 nM, respectively). Furthermore, glucose stimulated calcineurin phosphatase activity in mouse pancreatic islets, further supporting the view that calcineurin phosphatase activity is an essential part of glucose signaling to the human insulin gene. The high potency of cyclosporin A and tacrolimus in normal islets suggests that inhibition of insulin gene transcription by cyclosporin A and tacrolimus is clinically important and is one mechanism of the diabetogenic effect of these immunosuppressive drugs.Cyclosporin A and tacrolimus (also known as FK506) are powerful and clinically important immunosuppressive drugs that are widely used to prevent organ rejection after transplantation. In addition, an increasing number of autoimmune diseases are treated with these drugs. Both structurally distinct drugs bind to their respective intracellular receptors, the immunophilins, and the drug-immunophilin complexes then bind to and inhibit calcineurin phosphatase (Ho et al., 1996). Inhibition of calcineurin prevents the dephosphorylation of the nuclear factor of activated T cells (NFAT) and its nuclear translocation, resulting in decreased transcription of NFAT-dependent genes. Additional targets of calcineurin, such as the transcription factor cAMP response element-binding protein CREB (Schwaninger et al., 1993(Schwaninger et al., , 1995Barton et al., 1996;Krü ger et al., 1997), are likely to be involved. In this way, cyclosporin A and tacrolimus block early steps in T-cell activation.Among the most serious adverse effects of cyclosporin A and tacrolimus is an impaired glucose tolerance leading to hyperglycemia and diabetes mellitus (Kahan, 1989;Docherty and Clark, 1994;European FK506 Multicentre Liver Study

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“…One unit of the enzyme was defined as the amount of enzyme required to catalyze the release of 1 mol of phosphate/min. The activity of calcineurin was measured as described previously (42).…”

Section: Methodsmentioning

confidence: 99%

Usage of Tautomycetin, a Novel Inhibitor of Protein Phosphatase 1 (PP1), Reveals That PP1 Is a Positive Regulator of Raf-1 in Vivo

Mitsuhashi¹,

Shima²,

Tanuma³

et al. 2003

Journal of Biological Chemistry

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Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 M TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.Protein phosphatases regulate numerous cellular functions and signal transduction pathways in cooperation with protein kinases (1, 2). Protein phosphatase types 1 and 2A, known as PP1 1 and PP2A, are two of four major protein serine/threonine phosphatases (PPs) that regulate diverse cellular events such as cell division, transcription, translation, muscle contraction, glycogen synthesis, and neuronal signaling (3-5).Okadaic acid (OA), a polyether fatty acid from the marine black sponge Halichondria okadai, was first identified as a small molecular weight inhibitor of PP and has been studied extensively (6). More than 40 compounds that inhibit PP1 as well as PP2A have been identified. Using these natural compounds, numerous experiments have been performed to analyze the roles of PPs in various cellular events (6, 7). The IC 50 values of such phosphatase inhibitors are almost identical for PP1 and PP2A, with the exception of compounds such as OA, TF-23A, and fostriecin (8 -10). PP2A is selectively inhibited by OA, TF-23A, and fostriecin, and this selectivity has made it possible to analyze PP2A function in living cells. However, no known inhibitor inhibits PP1 specifically. Oikawa et al. (11) reported the total chemical synthesis of tautomycin (TM), a small molecular weight PP inhibitor originally isolated from Streptomyces spiroverticillatus. Using the synthesized TM and related compounds, we previously examined the structure-function relationship of TM and found that the left-and right-hand moieties of TM are required for inhibition of PP and induction of apoptosis, respectively (12). We also re...

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Development of an Assay Method for Activities of Serine/Threonine Protein Phosphatase Type 2B (Calcineurin) in Crude Extracts

Cited by 19 publications

References 37 publications

Immunophyllin Ligands Show Differential Effects on Alcohol Self-Administration in C57BL Mice

Immunophyllin Ligands Show Differential Effects on Alcohol Self-Administration in C57BL Mice

Inhibition of Human Insulin Gene Transcription by the Immunosuppressive Drugs Cyclosporin A and Tacrolimus in Primary, Mature Islets of Transgenic Mice

Usage of Tautomycetin, a Novel Inhibitor of Protein Phosphatase 1 (PP1), Reveals That PP1 Is a Positive Regulator of Raf-1 in Vivo

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