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)

The different specific inhibitors for phosphoserine/threonine and phosphotyrosine protein phosphatases were used to study the role of these protein phosphatases in collagen-platelet interaction. The collagen-induced platelet aggregation and the release reaction as measured ATP release were inhibited in a dose-dependent fashion by the addition of okadaic acid, a specific inhibitor of phosphoserine/threonine protein phosphatase 1 and 2A. The inhibition was also observed by the addition of a phosphotyrosine protein phosphatase inhibitor, vanadate. Suboptimal concentrations of both inhibitors together also inhibited collagen-induced platelet aggregation and release reaction in a concentration-dependent fashion. These results suggest that collagen-platelet interaction is modulated by both protein phosphatases.
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PMID:Okadaic acid and vanadate inhibit collagen-induced platelet aggregation; the functional relation of phosphatases on platelet aggregation. 138 63

We recently proposed a hypothesis for the molecular mechanism of the osteogenic action of fluoride in which it stimulates osteoblast proliferation via the inhibition of an osteoblastic acid phosphatase-like phosphotyrosyl protein phosphatase activity. To test this hypothesis, we investigated whether orthovanadate, a known phosphotyrosyl protein phosphatase inhibitor, would mimic fluoride in the stimulation of bone cell proliferation and bone collagen synthesis in vitro. Orthovanadate inhibited the osteoblastic acid phosphatase activity and stimulated bone cell proliferation at the same low concentrations (i.e. 5-15 microM). At the mitogenic doses, orthovanadate also showed a dose-dependent increase in alkaline phosphatase (a marker of mature osteoblasts) in cultured calvarial cells and stimulated bone collagen synthesis, as measured by the incorporation of [3H]proline and the conversion into [3H] hydroxyproline in organ calvaria cultures. Therefore, orthovanadate stimulated bone formation by increasing the number of mature osteoblasts mediated via stimulation of cell proliferation and differentiation. Orthovanadate was dependent on the presence of a mitogen in cell medium for its mitogenic action in vitro and synergistically potentiated the mitogenic actions on osteoblasts of those growth factors, i.e. insulin, epidermal growth factor, insulin-like growth factor I, and skeletal growth factor, whose mitogenic action involved tyrosyl protein phosphorylation. However, the interaction between orthovanadate and basic fibroblast growth factor, a growth factor that does not appear to involve tyrosyl protein phosphorylation, on bone cell proliferation was additive. In summary, these data are consistent with the hypothesis that inhibition of the osteoblastic phosphotyrosyl protein phosphatases can prolong and/or potentiate the mitogenic actions of growth factors, and thereby stimulates cell proliferation.
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PMID:Vanadate stimulates bone cell proliferation and bone collagen synthesis in vitro. 305 61

Contraction of intraocular fibrous membranes is an important feature in the pathogenesis of retinal detachment in proliferative vitreoretinopathy (PVR). Collagen gel contraction is a useful in vitro model of membrane contraction in PVR. We studied the role of protein kinase C (PKC) in collagen gel contraction induced by bovine choroidal fibroblasts and retinal pigment epithelial (RPE) cells. Collagen gels embedded with the cells were formed in culture dishes and gel contraction was evaluated. The PKC stimulator, phorbol 12-myristate 13-acetate (PMA), and the protein phosphatase 1 and 2A inhibitor, okadaic acid (OA), were used to evaluate the role of the PKC-mediated phosphorylation system in this gel contraction. Fifteen min incubation with PMA stimulated gel contraction, but 180 min incubation had no effect. Choroidal fibroblast- but not RPE cell-induced gel contraction was stimulated by OA. These effects were inhibited by the broad spectrum protein kinase inhibitor staurosporine and the specific PKC antagonist calphostin C. Transforming growth factor-beta (TGF-beta)1 and TGF-beta 2, which are known to be present in eyes with PVR, were evaluated to determine their effect on gel contraction. Both TGF-beta 1 and 2 had a stimulatory effect on contraction of gels seeded with choroidal fibroblasts and RPE cells, but staurosporine and calphostin C inhibited this TGF-beta-induced gel contraction. These results indicate that activation of PKC/protein phosphorylation is an important factor in gel contraction caused by choroidal fibroblasts and RPE cells, and that TGF-beta-induced gel contraction is mediated at least in part via the PKC pathway.
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PMID:Collagen gel contraction induced by retinal pigment epithelial cells and choroidal fibroblasts involves the protein kinase C pathway. 792 9

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

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.
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PMID:The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane. 816 55

Okadaic acid, a specific inhibitor of phosphoserine/threonine protein phosphatases 1 and 2A, was used to determine whether these protein phosphatases play a role in collagen-induced platelet aggregation and release reaction as measured by ATP release. Collagen-induced platelet aggregation and ATP release were inhibited by the addition of okadaic acid to platelet-rich plasma in a dose-dependent manner. The inhibitory effect of okadaic acid on collagen-induced platelet aggregation correlated with phosphorylation of proteins with M(r) 14.4, 25, 32, 36, 50, 60, and 80 kDa. The 14.4-kDa protein was purified to apparent homogeneity by electroelution from gel slices. This protein reacted with antibodies to phospholipase A2 (PLA2). Since okadaic acid inhibited PLA2 activity in platelet-rich plasma but not in the PLA2 assay mixture, the effect appears to be indirect. Furthermore, using a combination of immunoprecipitation and measurement of enzyme activity, PLA2 activity was inhibited in the presence of okadaic acid. The inhibited activity could not be restored by the addition of collagen. These results suggest that the phosphorylated form of PLA2 is inactive. Using [32P]glycogen phosphorylase a as substrate, protein phosphatase activity was inhibited by okadaic acid in a concentration-dependent manner. An immunoblot of platelet homogenates with anti-protein phosphatase 1 showed a band with M(r) 50 kDa reacting with the antibodies, suggesting that the 50-kDa protein is protein phosphatase 1. These data clearly show that okadaic acid increases the phosphorylation and indirectly decreases the activity of PLA2, but whether inhibition of PLA2 activity is related to collagen-induced platelet aggregation and release reaction remains to be determined.
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PMID:The role of protein phosphatases 1 and 2A in collagen-platelet interaction. 838 5

MDCK cells cultured in Type I collagen gels can be induced to develop branching tubular structures with demonstrable lumens in the presence of hepatocyte growth factor (HGF). As we have now shown by immunofluorescent localization of specific marker proteins, these tubules retain apical-basolateral polarity. However, the secondary signaling events which lead to these characteristic morphogenetic changes induced by HGF remain largely unelucidated. In order to examine these signaling events, particularly the role of protein phosphorylation in the formation of branching tubular structures, Madin-Darby canine kidney (MDCK) cells in collagen gels were treated with HGF plus well-characterized agents that affect protein phosphorylation. We quantified the formation of branching processes, an early step in the development of tubular structures in this model. Protein kinase C (PKC) inhibition resulted in more complex branching processes in the presence of HGF, when compared with HGF alone. In contrast, treatment with activators of protein kinase A (PKA), as well as calmodulin antagonists, caused a marked decline in process formation. Consistent with an important role for protein phosphorylation in HGF-induced morphogenesis, protein phosphatase inhibition by okadaic acid or calyculin A was found to markedly inhibit process formation. Tyrosine kinase (TK) inhibition also decreased the percentage of processes. This is consistent with data indicating that one of the HGF receptors is identical to the c-met protooncogene product, which is known to possess TK activity. Our results suggest that the HGF-mediated induction of branching processes in MDCK cells, an early step in the development of branching tubular structures, can be modulated by multiple phosphorylation mechanisms including those mediated by PKC, PKA, and Ca2+/calmodulin-dependent kinase(s). We discuss how these phosphorylation events may play crucial roles in determining the degree of tubule formation and their length, as well as the extent of their arborization during the early development of epithelial tissues.
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PMID:Modulation of HGF-induced tubulogenesis and branching by multiple phosphorylation mechanisms. 840 77

Transforming growth factor beta 1 (TGF-beta 1) exerts a positive effect on the transcription of genes coding for several extracellular matrix-related products, including collagen I. We have previously identified a strong TGF-beta 1-responsive element (TbRE) in the upstream promoter sequence of the alpha 2(I) collagen (COL1A2) gene. Our experiments have shown that TGF-beta 1 stimulates COL1A2 transcription by increasing binding of an Sp1-containing complex (TbRC) to the TbRE. They have also suggested that the change occurs via posttranslational modification of a protein(s) directly or indirectly interacting with Sp1. Here, we provide evidence showing that tyrosine dephosphorylation of nuclear proteins mimics the stimulation of COL1A2 transcription by the TGF-beta 1-activated signaling pathway. Preincubation of nuclear extracts with protein tyrosine phosphatase (PTPase) but not with protein phosphatase type 2A (PP2A), a serine/threonine phosphatase, enhanced binding of the TbRC to the same degree as culturing cells in TGF-beta 1. Consistent with these in vitro findings, genistein, a tyrosine kinase inhibitor, led to markedly increased COL1A2 gene expression, whereas sodium orthovanadate, a tyrosine phosphatase inhibitor, decreased it substantially. These results were supported by transfection experiments showing that genistein and sodium orthovanadate have opposite effects on TbRE-mediated transcription. Moreover, nuclear proteins isolated from genistein-treated cells were found to interact with the TbRE significantly more than those from untreated cells. Furthermore, pretreatment of cells with sodium orthovanadate virtually abrogated nuclear protein binding to the TbRE, but not to a neighboring cis-acting element unresponsive to TGF-beta 1. The results of this study, therefore, provide the first correlation between tyrosine dephosphorylation, increased binding of a transcriptional complex, and TGF-beta 1 stimulation of gene expression.
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PMID:Tyrosine dephosphorylation of nuclear proteins mimics transforming growth factor beta 1 stimulation of alpha 2(I) collagen gene expression. 852 47

Differentiating chick limb-bud mesenchymal cells plated in micromass culture form a cartilage matrix that can be mineralized in the presence of 4 mM inorganic phosphate (Pi), and 1 mM calcium. Previous studies showed that when beta-glycerophosphate (beta GP) is used in place of Pi, the mineral crystals formed are larger and differ in distribution. The present study shows that the difference in distribution is not associated with alterations in cell proliferation, protein synthesis, or with collagen, proteoglycan core protein, or alkaline phosphatase gene expression. Cultures with 2.5, 5, and 10 mM beta GP did show different levels of alkaline phosphatase activity, and in the presence of low (0.3 mM) Ca had different Pi contents (4, 6 and 9 mM, respectively), indicating that the increase in CaxP product may in part be responsible for the altered pattern of mineralization. However, cultures with beta GP in which alkaline phosphatase activity was inhibited with levamisole still had an altered mineral distribution as revealed by Fourier transform-infrared (FT-IR) microspectroscopy. The presence of a casein kinase II-like activity in the mineralizing cultures, the ability of specific inhibitors of this enzyme to block mineralization, and the known ability of beta GP to block phosphoprotein phosphatase activity suggests that altered patterns of matrix protein phosphorylation may influence mineral deposition in these cultures.
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PMID:The mechanism of beta-glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures. 891 77

The interstitial collagenase produced by the rat growth plate chondrocytes is the homologue of the human collagenase-3, or matrix metalloproteinase-13. This enzyme is responsible for the loss of collagen during hypertrophy of chondrocytes and for the degradation of transverse septa in long bone growth. Rachitic rats (42 days, male Sprague-Dawley) had an 8-fold higher level of collagenase mRNA in the hypertrophic versus proliferative zone of growth plate cartilage. Intramuscular injection of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; 1.0 micrograms/kg body weight) in rachitic rats increased collagenase mRNA another 1.5-fold in the hypertrophic zone. The regulation of collagenase gene by 1,25-(OH)2D3 and interleukin (IL)-1 beta in cultured proliferative chondrocytes was studied by means of steady-state mRNA and half-life determination of mRNA using the transcriptional inhibitor actinomycin D, and nuclear run-on transcription analyses. Treatment of cells with 1,25-(OH)2D3 (10(-6) M) and IL-1 beta (2 ng/ml) increased collagenase mRNA 8- and 13-fold, respectively. Additionally, the collagenase mRNA half-life was increased by 1,25-(OH)2D3 and IL-1 beta. In the presence of a protein kinase C inhibitor, staurosporine, 1,25-(OH)2 D3 induction of collagenase mRNA was blocked. Here the addition of phorbol 12-myrisate 13-acetate (PMA) to activate protein kinase C increased collagenase mRNA 10-fold. However, in the presence of staurosporine (50 nM), PMA induction was blocked, whereas IL-1 beta was not. IL-1 beta is known to activate several phosphorylation pathways. Okadaic acid (500 nM), a protein phosphatase inhibitor, increased the relative collagenase mRNA abundance 10-fold. The rate of the rat collagenase gene transcription in nuclei was increased with 1,25-(OH)2D3, IL-1 beta and okadaic acid. In separate experiments, the collagenase promoter was ligated to a reporter plasmid and the plasmid was transfected into chondrocytes. The results showed that 1,25-(OH)2D3, IL-1 beta, and PMA increased reporter activity 2.5-, 2.8-, and 3.27-fold, respectively. Thus, there are multiple nuclear and cytoplasmic mechanisms by which cartilage modulators regulate rat interstitial collagenase gene expression.
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PMID:Regulation of rat interstitial collagenase gene expression in growth cartilage and chondrocytes by vitamin D3, interleukin-1 beta, and okadaic acid. 897 56


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