Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An aortic phosphatase which dephosphorylates several proteins including phosphorylase a and the 20-kDa myosin light chains is subject to modulation in vitro by polycationic effectors such as lysine-rich histone-H1 and polylysine. This study was based on the hypothesis that polycationic modulation of expressed enzymic activity involves interactions between the effectors and a regulatory site associated with the polycation-modulated (PCM)-phosphatase. Basal PCM-phosphatase activity expressed against myocardial myosin light chains (MLC, 1258 nmole/min/mg) was about eightfold greater than activity expressed against phosphorylase a (149 nmole/min/mg). However, dephosphorylation of phosphorylase a was stimulated four- to sevenfold by low concentrations of polylysine (Mr = 13,000; 0.01-0.1 microM), whereas MLC phosphatase activity was virtually abolished. Higher concentrations of polylysine inhibited dephosphorylation of either substrate. Interestingly, a heat-stable fraction prepared from the PCM-phosphatase reversed the stimulatory effect of polylysine on phosphorylase phosphatase activity and the inhibitory effect on dephosphorylation of MLC. No reversal of the modulatory effects of polylysine occurred when protein phosphatase inhibitor 1 or inhibitor 2 was substituted for the heat-stable factor derived from the PCM-phosphatase. Sucrose density centrifugation of the enzyme yielded a single peak (Mr = 63,000) exhibiting polycation-modulated activity against phosphorylase a and MLC. Moreover, heating each of the gradient fractions showed the presence of a heat-stable factor which reversed the modulatory effects of polylysine on dephosphorylation of either phosphorylase a or MLC. These results show that a specific heat-stable factor, which differs from both inhibitor 1 and 2, is associated with the PCM-phosphatase. The results suggest that polycationic modulation of expressed PCM-phosphatase activity may involve interactions between the polycationic effector and the enzyme-associated regulatory factor.
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PMID:A new heat-stable regulatory factor is associated with aortic polycation-modulated (PCM)-phosphatase. 300 44

Two potent inhibitors of protein phosphatase type 1 (PP1) and type 2A (PP2A), calyculin A (CAL-A) and okadaic acid (OKA), inhibited human platelet aggregation induced by thrombin, collagen and 9,11-epithio-11,12-methano-thromboxane A2 (STA2). IC50 values of CAL-A and OKA for STA2-induced aggregation were 53 nM and 3.5 microM, respectively. These drugs also inhibited thrombin-induced [14C]serotonin secretion of platelets. CAL-A and OKA elicited phosphorylation of certain proteins with an apparent M(r) (x 10(-3) of 200, 60, 50 and 20 light chain of myosin (MLC). Agonist-induced 47,000 M(r) protein phosphorylation was strongly inhibited by these compounds, whereas phosphorylation of 20,000 M(r) MLC was enhanced. The increase in 50,000 M(r) protein phosphorylation by CAL-A and OKA was observed in the presence of agonists, and the 50,000 M(r) phosphorylation may be involved in the inhibition of platelet activation by these compounds. Subcellular analysis of the phosphatase activity in human platelets showed that MLC phosphatase activity was present mainly (approx. 78%) in the cytosolic fraction. Chromatography of human platelet extract on heparin-Sepharose resolved two peaks of MLC phosphatase activity: PP2A in 0.1 M NaCl eluate and PP1 in 0.5 NaCl eluate. PP2A and PP1 isozymes (PP1 alpha, PP1 gamma and PP1 delta) have also been identified in human platelets, by cross-reactivity with polyclonal antibodies against PP2A and PP1 isozymes, respectively. These results suggest that PP1 and/or PP2A may play an important role in the process of platelet activation by regulating levels of phosphorylation of certain proteins.
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PMID:Calyculin A and okadiac acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A. 801 29

Upon platelet activation by a high shear stress (108 dyne/cm2), actin and actin-binding protein increased rapidly into the Triton-insoluble cytoskeleton, whereas the association of myosin increased gradually. The amounts of cytoskeleton-associated myosin depended on the extent of aggregation. Preceding the maximal aggregation and ATP secretion, the 20 kDa light chain of myosin (MLC) is rapidly phosphorylated to approx. 45% of 20 kDa MLC and is then dephosphorylated. Cytoskeletal association of myosin and phosphorylation of 20 kDa MLC was inhibited by OP-41483, a prostaglandin I2 analog, which inhibited the full aggregation response to shear stress. Exposure to high shear stress resulted in an increased association of myosin light chain kinase and protein phosphatase types 1 and 2A with the cytoskeleton, while the cytoskeletal association of protein kinase C was not evident. These results indicate that 20 kDa MLC phosphorylation is involved in shear stress-induced platelet activation, and that cytoskeletal association of protein phosphatases may regulate the phosphorylation level of cytoskeletal elements such as myosin together with myosin light chain kinase.
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PMID:Shear stress-induced myosin association with cytoskeleton and phosphorylation in human platelets. 907 28

We have recently shown that several putative selective inhibitors of Ca2+-calmodulin-dependent myosin light chain kinase (MLCK), such as ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], reversibly stimulate renin secretion [C. S. Park, S.-H. Chang, H. S. Lee, S.-H. Kim, J. W. Chang, and C. D. Hong. Am. J. Physiol. 271 (Cell Physiol. 40): C242-C247, 1996]. We hypothesized that Ca2+ inhibits renin secretion, via phosphorylation of 20-kDa myosin light chain (MLC20), by activating MLCK. In the present studies, we have investigated the types of protein phosphatase (PP) involved in the control of renin secretion through inhibition of MLC dephosphorylation using inhibitors of various types of serine/threonine-specific protein phosphatases. Cyclosporin A, a putative inhibitor of PP type 2 (calcineurin), was without effect. Calyculin A and okadaic acid, putative selective inhibitors of both PP type 1 (PP1) and type 2A (PP2A), significantly inhibited renin secretion under control conditions. Calyculin A had inhibitory effects at least 10-fold more potent than okadaic acid, suggesting that PP1, rather than PP2A, is involved in the control of renin secretion. Furthermore, calyculin A blocked the reversal of renin secretion preinhibited by raised intracellular Ca2+ concentrations in a concentration-dependent manner. Calyculin A (10(-6) M) significantly inhibited renin secretion stimulated by lowering intracellular Ca2+ concentrations and blocked the stimulatory effect of ML-9 on renin secretion. Taking all of these results into consideration, we hypothesize that dephosphorylation of MLC20 by Ca2+-independent PP1 stimulates renin secretion, whereas phosphorylation of MLC20 by Ca2+-calmodulin-dependent MLCK inhibits it. This hypothesized regulatory model of renin secretion predicts that the rate of renin secretion at a given time is determined by the ratio of phosphorylated to dephosphorylated MLC20, which is, in turn, determined by the dynamic balance between activity of MLCK and MLC phosphatase.
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PMID:Inhibitory effect of calyculin A, a Ser/Thr protein phosphatase type I inhibitor, on renin secretion. 981 25

The cellular events leading to cerebral vasospasm after subarachnoid haemorrhage are poorly understood, although an increase in smooth muscle myosin light chain phosphorylation has been observed. This study set out to determine if phosphatase inhibition may be involved in the pathological maintenance of tension observed during vasospasm. We found that 1 nM okadaic acid, a type 2A protein phosphatase inhibitor, elicited an increase in rate of O(2) consumption in the porcine carotid artery similar to that by cerebrospinal fluid (CSF) from vasospastic patients (CSF(V), n=5) (control 0.23+/-0.03, CSF(V) 0.84+/-0.16 and okadaic acid 0.85+/-0.02 micromol min(-1) g dwt(-1)). It was also observed that phosphatase inhibition with 1 nM okadaic acid significantly slowed relaxation after a stretch in a similar fashion to CSF(V) haemorrhage. CSF from vasospastic subarachnoid haemorrhage patients, but not from those without vasospasm, contains an extractable substance which modulates myosin light chain phosphorylation in vitro. A phosphatase preparation obtained from the porcine carotid artery dephosphorylated 63+/-2% of the phosphorylated (MLC(20)) substrate in vitro, and non-vasospastic CSF treated enzyme dephosphorylated 60+/-2.6%. Okadaic acid inhibited phosphatase dephosphorylated only 7.5+/-1% of the substrate where CSF(V) treated enzyme dephosphorylated 22+/-2.8% of the substrate. We conclude that inhibition of smooth muscle phosphatase may be involved in the mechanisms associated with cerebral vasospasm after subarachnoid haemorrhage.
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PMID:The presence of an extractable substance in the CSF of humans with cerebral vasospasm after subarachnoid haemorrhage that correlates with phosphatase inhibition. 1077 79

The serine/threonine phosphatase calcineurin is an important regulator of calcium-activated intracellular responses in eukaryotic cells. In higher eukaryotes, calcium/calmodulin-mediated activation of calcineurin facilitates direct dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). Recently, controversy has surrounded the role of calcineurin in mediating skeletal muscle cell hypertrophy. Here we examined the ability of calcineurin-deficient mice to undergo skeletal muscle hypertrophic growth following mechanical overload (MOV) stimulation or insulin-like growth factor-1 (IGF-1) stimulation. Two distinct models of calcineurin deficiency were employed: calcineurin Abeta gene-targeted mice, which show a approximately 50% reduction in total calcineurin, and calcineurin B1-LoxP-targeted mice crossed with a myosin light chain 1f cre knock-in allele, which show a greater than 80% loss of total calcineurin only in skeletal muscle. Calcineurin Abeta-/- and calcineurin B1-LoxP(fl/fl)-MLC-cre mice show essentially no defects in muscle growth in response to IGF-1 treatment or MOV stimulation, although calcineurin Abeta-/- mice show a basal defect in total fiber number in the plantaris and a mild secondary reduction in growth, consistent with a developmental defect in myogenesis. Both groups of gene-targeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overload-mediated fiber-type switching was dramatically impaired in calcineurin B1-LoxP(fl/fl)-MLC-cre mice. NFAT-luciferase reporter transgenic mice failed to show a correlation between IGF-1- or MOV-induced hypertrophy and calcineurin-NFAT-dependent signaling in vivo. We conclude that calcineurin expression is important during myogenesis and fiber-type switching, but not for muscle growth in response to hypertrophic stimuli.
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PMID:Genetic loss of calcineurin blocks mechanical overload-induced skeletal muscle fiber type switching but not hypertrophy. 1508 23

We analyzed the signaling pathways initiated by endothelin receptors ETA and ETB in intestinal circular and longitudinal smooth muscle cells. The response to endothelin-1 (ET-1) consisted of two phases in both cell types. The initial, transient phase of contraction and phosphorylation of 20-kDa myosin light chain (MLC20) was mediated additively by ETA and ETB receptors and initiated by Galphaq-, Ca2+/calmodulin-dependent activation of MLC kinase. In contrast, the sustained phase was mediated selectively by ETA receptors via a pathway involving sequential activation of Galpha13, RhoA, and Rho kinase, resulting in phosphorylation of MYPT1 at Thr696 and phosphorylation of MLC20. Although PKC was activated, CPI-17 was not phosphorylated and hence did not contribute to inhibition of MLC phosphatase. The absence of CPI-17 phosphorylation by PKC reflected active dephosphorylation of CPI-17 by protein phosphatase 2A (PP2A). PP2A was activated via a pathway involving ETB-dependent stimulation of p38 MAPK activity. CPI-17 phosphorylation was unmasked in the presence of the ETB antagonist BQ-788, but not the ETA antagonist BQ-123, and in the presence of a low concentration of okadaic acid, which selectively inactivates PP2A. The resultant phosphorylation of CPI-17 was blocked by bisindolylmaleimide, providing direct confirmation that it was PKC dependent. We conclude that the two phases of the intestinal smooth muscle response to ET-1 involve distinct receptors, G proteins, and signaling pathways. The sustained response is mediated via selective ETA-dependent phosphorylation of MYPT1. In contrast, ETB initiates an inhibitory pathway involving p38 MAPK-dependent activation of PP2A that causes dephosphorylation of CPI-17.
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PMID:Gq/G13 signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ETA and CPI-17 dephosphorylation via ETB. 1547 16

We investigated the development of donor antigen-specific hyporeactivity by using donor cells as stimulator cells in the MLC and comparing the pre- and post-transplant responses of peripheral blood mononuclear cells. Twenty-two haploidentical pediatric living-relative donor recipients treated with daclizumab, methylprednisone, mofetil mycophenolate and calcineurin inhibitors were tested for study. Of these, 50% of the recipients developed in vitro donor antigen-specific hyporeactivity. The recipients who did so have higher creatinine clearance levels at 12, 24 and 36 months post-transplant (104, 92 and 81 mL/min/1.73 m(2), respectively) than those who remained responsive to donor antigens (77, 74 and 70 mL/min/1.73 m(2)) (p < 0.05). Acute rejection episodes were not observed; however, no recipients with donor-specific hyporeactivity have been diagnosed with CAN, unlike three recipients who remained responsive to donor antigens (0% vs. 27.3%, p = 0.06). Differences in accumulative doses of methylprednisone and mofetil mycophenolate were observed between hyporeactivity- and response-patients to donor antigens at the three years end-point (1.9 +/- 0.8 g/m(2) vs. 4.2 +/- 0.5 g/m(2), and 277 +/- 89 g/m(2) vs. 672 +/- 16.0 g/m(2); p < 0.01 and <0.02, respectively). We conclude that the development of donor antigen-specific hyporeactivity correlate with improved graft function and may permit lower immunosuppression.
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PMID:Improved long-term graft function in kidney transplant recipients with donor antigen-specific hyporeactivity. 1730 Apr 92

Platelets are produced by megakaryocytes (MKs) through proplatelet formation (PPF), or cytoplasmic extensions, in vitro. Through the use of video-enhanced light microscopy, as well as localization of cytoskeletal proteins by confocal microscopy, the reaction of fully mature MK proplatelets, derived from murine embryonic stem cells, to various agents was studied. Calyculin A (protein phosphatase 1/2A inhibitor) treatment induced proplatelet retraction. In MKs with PPF, the expression of actin, myosin IIA, monophosphorylated myosin light chain (MLC-P1), and diphosphorylated myosin light chain (MLC-P2) was diffusely located. Following calyculin A treatment, actin was diffusely localized in retracted MKs and was expressed particularly in the periphery. MLC-P1 was also localized primarily in the periphery; however, MLC-P2 was expressed mostly in the inner area of proplatelets. Protein phosphatase inhibitors may result in increased hyperphosphorylation of localized MLC, which could alter the balance of actomyosin force in a cell, and therefore induce proplatelets retraction.
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PMID:Calyculin A retraction of mature megakaryocytes proplatelets from embryonic stem cells. 1808 16

Myostatin (Mstn) is a secreted growth factor belonging to the tranforming growth factor (TGF)-beta superfamily. Inactivation of murine Mstn by gene targeting, or natural mutation of bovine or human Mstn, induces the double muscling (DM) phenotype. In DM cattle, Mstn deficiency increases fast glycolytic (type IIB) fiber formation in the biceps femoris (BF) muscle. Using Mstn null ((-/-)) mice, we suggest a possible mechanism behind Mstn-mediated fiber-type diversity. Histological analysis revealed increased type IIB fibers with a concomitant decrease in type IIA and type I fibers in the Mstn(-/-) tibialis anterior and BF muscle. Functional electrical stimulation of Mstn(-/-) BF revealed increased fatigue susceptibility, supporting increased type IIB fiber content. Given the role of myocyte enhancer factor 2 (MEF2) in oxidative type I fiber formation, MEF2 levels in Mstn(-/-) tissue were quantified. Results revealed reduced MEF2C protein in Mstn(-/-) muscle and myoblast nuclear extracts. Reduced MEF2-DNA complex was also observed in electrophoretic mobility-shift assay using Mstn(-/-) nuclear extracts. Furthermore, reduced expression of MEF2 downstream target genes MLC1F and calcineurin were found in Mstn(-/-) muscle. Conversely, Mstn addition was sufficient to directly upregulate MLC promoter-enhancer activity in cultured myoblasts. Since high MyoD levels are seen in fast fibers, we analyzed MyoD levels in the muscle. In contrast to MEF2C, MyoD levels were increased in Mstn(-/-) muscle. Together, these results suggest that while Mstn positively regulates MEF2C levels, it negatively regulates MyoD expression in muscle. We propose that Mstn could regulate fiber-type composition by regulating the expression of MEF2C and MyoD during myogenesis.
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PMID:Myostatin regulates fiber-type composition of skeletal muscle by regulating MEF2 and MyoD gene expression. 1912 64


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