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)

Raf-1 serine/threonine protein kinase plays an important role in cell survival, proliferation, and migration; however, the specific targets of Raf-1 in diverse cellular processes are not clearly defined. Myosin phosphatase activity is critical to the regulation of cytoskeletal reorganization, cytokinesis, and cell motility. Here, we describe the association of Raf-1 with myosin phosphatase and phosphorylation of the regulatory myosin-binding subunit (MBS) of myosin phosphatase by Raf-1. Treatment of cells with phorbol 12-myristate 13-acetate has been shown to stimulate Raf-1 protein kinase. To determine the effect of enzymatic activation of Raf-1 on MBS phosphorylation, COS-1 cells were transiently transfected with FLAG-tagged full-length Raf-1. A significantly higher phosphorylation of purified glutathione S-transferase-tagged truncated MBS protein (amino acids 654-880) occurred in the presence of FLAG-Raf-1 immunoprecipitated from phorbol 12-myristate 13-acetate-treated cells compared with untreated cells ( approximately 3.0-fold). Using a sequential kinase-phosphatase assay and phosphorylated myosin light chain as substrate in the phosphatase reaction, we showed that Raf-1-associated protein phosphatase-specific activity was inhibited (relative phosphatase activity without and with adenosine 5'-O-(3-thiotriphosphate): 100 and approximately 30%, respectively). Previously, ionizing radiation has been shown to activate Raf-1 (Kasid, U., Suy, S., Dent, P., Ray, S., Whiteside, T. L., and Sturgill, T. W. (1996) Nature 382, 813-816). Exposure of cells to ionizing radiation resulted in the increased association of Raf-1 with MBS (3-6-fold versus unirradiated control) and inhibition of Raf-1-associated protein phosphatase-specific activity (relative phosphatase activity without and with ionizing radiation: 100 and approximately 54%, respectively). Our studies identify MBS as a new substrate of Raf-1 and implicate a role for Raf-1 in the regulation of pathways involving myosin phosphatase activity.
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PMID:Phosphorylation of the myosin-binding subunit of myosin phosphatase by Raf-1 and inhibition of phosphatase activity. 1171 7

FKBP51 is a member of the immunophilin family having intrinsic peptidyl-prolyl cis-trans-isomerase (PPIase) activity. Its enzymatic activity is inhibited by binding either immunosuppressive agent FK506 or rapamycin. Similar to FKBP12, but at higher concentrations of FK506, FKBP51 has been shown to inhibit the serine/threonine phosphatase activity of calcineurin in the presence of calcium and calmodulin. Here we show that a glutathione S-transferase (GST) fusion protein of FKBP51 on glutathione-Sepharose beads precipitated both purified calcineurin from bovine brain and calcineurin from murine T cell lysates. Surprisingly, the binding of GST-FKBP51 to calcineurin was FK506-independent and independent of a requirement for calcium or exogenous calmodulin. Unlike FKBP12, FKBP51 transiently expressed in COS-7 cells was precipitated by calcineurin bound to calmodulin-Sepharose beads in the absence of either FK506 or rapamycin. Unlike FKBP12, however, overexpression of FKBP51 in Jurkat T cells did not significantly affect the transcriptional activation of nuclear factor of activated T cells (NFAT) upon physiological stimulation, nor did it affect the ability of FK506 to inhibit NFAT-driven transcription. We generated a series of FKBP51 mutations to map the interaction of FKBP51 with calcineurin. Deletion of the aminoterminal, FKBP12-like domain of FKBP51 did not affect the ability of FKBP51 to bind to purified calcineurin, while deletion of the FKBP51 carboxyterminal domain abrogated the ability of FKBP51 to bind to calcineurin. Taken together, these results demonstrate a novel interaction between calcineurin and the immunophilin FKBP51 that is independent of calcium, calmodulin, and drug. The binding site on calcineurin for FKBP51 is separable from the immunophilin PPIase-active and drug-binding site.
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PMID:Calcium- and FK506-independent interaction between the immunophilin FKBP51 and calcineurin. 1181 52

When UMR-106 osteoblastic cells, LLCPK1 kidney cells, and VDR transfected COS-7 cells were transfected with the rat 24-hydroxylase [24(OH)ase] promoter (-1,367/+74) or the mouse osteopontin (OPN) promoter (-777/+79), we found that the response to 1,25dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] could be significantly enhanced 2- to 5-fold by the protein phosphatase inhibitor, okadaic acid (OA). Enhancement of 1,25-(OH)(2)D(3)-induced transcription by OA was also observed using a synthetic reporter gene containing either the proximal 24(OH)ase vitamin D response element (VDRE) or the OPN VDRE, suggesting that the VDRE is sufficient to mediate this effect. OA also enhanced the 1,25-(OH)(2)D(3)-induced levels of 24(OH)ase and OPN mRNA in UMR osteoblastic cells. The effect of OA was not due to an up-regulation of VDR or to an increase in VDR-RXR interaction with the VDRE. To determine whether phosphorylation regulates VDR-mediated transcription by modulating interactions with protein partners, we examined the effect of phosphorylation on the protein-protein interaction between VDR and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex, using glutathione-S-transferase pull-down assays. Similar to the functional studies, OA treatment was consistently found to enhance the interaction of VDR with DRIP205 3- to 4-fold above the interaction observed in the presence of 1,25-(OH)(2)D(3) alone. In addition, studies were done with the activation function-2 defective VDR mutant, L417S, which is unable to stimulate transcription in response to 1,25-(OH)(2)D(3) or to interact with DRIP205. However, in the presence of OA, the mutant VDR was able to activate 24(OH)ase and OPN transcription and to recruit DRIP205, suggesting that OA treatment may result in a conformational change in the activation function-2 defective mutant that creates an active interaction surface with DRIP205. Taken together, these findings suggest that increased interaction between VDR and coactivators such as DRIP205 may be a major mechanism that couples extracellular signals to vitamin D action.
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PMID:Enhancement of VDR-mediated transcription by phosphorylation: correlation with increased interaction between the VDR and DRIP205, a subunit of the VDR-interacting protein coactivator complex. 1181 2

Glycogen synthase kinase-3 (GSK-3) is regulated by various extracellular ligands and phosphorylates many substrates, thereby regulating cellular functions. Using yeast two-hybrid screening, we found that GSK-3beta binds to AKAP220, which is known to act as an A-kinase anchoring protein. GSK-3beta formed a complex with AKAP220 in intact cells at the endogenous level. Cyclic AMP-dependent protein kinase (PKA) and type 1 protein phosphatase (PP1) were also detected in this complex, suggesting that AKAP220, GSK-3beta, PKA, and PP1 form a quaternary complex. It has been reported that PKA phosphorylates GSK-3beta, thereby decreasing its activity. When COS cells were treated with dibutyryl cyclic AMP to activate PKA, the activity of GSK-3beta bound to AKAP220 decreased more markedly than the total GSK-3beta activity. Calyculin A, a protein phosphatase inhibitor, also inhibited the activity of GSK-3beta bound to AKAP220 more strongly than the total GSK-3beta activity. These results suggest that PKA and PP1 regulate the activity of GSK-3beta efficiently by forming a complex with AKAP220.
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PMID:A-kinase anchoring protein AKAP220 binds to glycogen synthase kinase-3beta (GSK-3beta ) and mediates protein kinase A-dependent inhibition of GSK-3beta. 1214 1

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 microm 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.
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PMID:Usage of tautomycetin, a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo. 1237 92

The endothelial isoform of nitric oxide synthase (eNOS) is a calcium/calmodulin-dependent enzyme that catalyzes the synthesis of nitric oxide, a key mediator of vascular homeostasis. eNOS undergoes a variety of posttranslational modifications, including phosphorylation on at least three residues: serines 116 and 1179 and threonine 497. Although the agonist-modulated protein kinase pathways that lead to eNOS phosphorylation have been studied in detail, the signaling pathways governing eNOS dephosphorylation remain less well characterized. The present study identifies protein phosphatase 2A (PP2A) as a key determinant of eNOS dephosphorylation and enzyme activity. We transfected bovine aortic endothelial cells (BAEC) with epitope-tagged cDNAs encoding wild-type eNOS or a series of phosphorylation-deficient eNOS mutants, immunoprecipitated [(32)P(i)] biosynthetically labeled recombinant proteins using antibodies directed against the epitope tag and treated the [(32)P(i)]-phosphorylated eNOS with protein phosphatases. We found that PP2A dephosphorylates eNOS residues threonine 497 and serine 1179 but not serine 116 and that an eNOS mutant lacking these three established phosphorylation sites is robustly labeled when expressed in BAEC and is dephosphorylated by PP2A. An inhibitor of PP2A increases eNOS enzymatic activity and augments overall levels of eNOS phosphorylation, specifically increasing phosphorylation of serines 116 and 1179. When transfected into BAEC or COS-7 cells, a "phospho-mimetic" eNOS mutant in which threonine 497 is changed to aspartate shows attenuated phosphorylation at serine 1179 as well as reduced enzyme activity in COS-7 cells. Our results indicate that regulation of eNOS dephosphorylation may be a key point for control of nitric oxide-dependent signaling pathways in vascular endothelial cells.
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PMID:Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites. 1250 Dec 14

Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein kinase A or C, the density of co-localizing Na,K-EGFP and transferrin vesicles was increased 3-4-fold, while the ouabain-sensitive 86Rb uptake was reduced by 22%. Simultaneous activation of PKA and PKC had additive effects with a 6-fold increase of co-localization and a 38% reduction of 86Rb uptake. Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation or protein phosphatase inhibition.
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PMID:Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C. 1253 74

BAD is a BH3-only protein, and its proapoptotic activity is negatively regulated by serine phosphorylation. Here, we show that overexpression of BAD preferentially augments anchorage loss-induced apoptosis (anoikis). Gene transfer-mediated BAD overexpression alone did not induce apoptosis in attached MDCK cells but strongly augmented apoptosis when cells were cultured in suspension. In contrast, overexpression of another BH3-only protein, BID, displayed much lower augmentation of anoikis, suggesting a preferential contribution of BAD to anoikis. During suspension culture, unphosphorylated BAD was gradually increased and targeted to the mitochondria. Cotransfection of BAD with constitutively active Akt cDNA strongly inhibited this change. In contrast, the increase of unphosphorylated BAD was not significantly inhibited by several phosphatase inhibitors or cotransfection with a dominant negative calcineurin cDNA, implying that the increase may be mainly due to a decrease of serine kinase activity, such as that of Akt. Similar results were observed in COS-7 cells, suggesting that BAD overexpression can increase sensitivity of anchorage-dependent cancer cells to anoikis. Thus, we propose that BAD can serve as a valuable gene therapeutic molecule to inhibit carcinoma progression.
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PMID:Overexpression of BAD preferentially augments anoikis. 1294 97

F10, a subline of the B16 mouse melanoma cell line, is itself the parent of the more metastatic BL6 line. BL6 cells differ from F10 cells by an alteration of the gene encoding the B56gamma regulatory subunit of protein phosphatase 2A (PP2A), which results in mRNA encoding a truncated variant of the subunit (deltagamma1). Expression of deltagamma 1 protein is detectable only when BL6 cells are transplanted into mice and then gamma-irradiated. Recently, B56gamma subunit-containing PP2A holoenzymes have shown to dephosphorylate Mdm2, a negative regulator of p53. Thus, we assessed whether the expression of deltagamma1 affects irradiation-induced phosphorylation of Mdm2 and radioresistance of melanoma cells by perturbing the regulation of p53. Western blot analyses revealed that irradiated COS-7 and NIH3T3 cells stably expressing deltagamma1 showed significantly less irradiation-induced Mdm2 phosphorylation. Mdm2 phosphorylation reduces the ability of Mdm2 to target p53 for degradation, which probably explained why p53 protein levels in NIH3T3 cells expressing deltagamma1 were not significantly elevated by irradiation, unlike in wild-type cells. This was also true for F10 cells transfected with deltagamma1 (F10deltagamma1) when the cells expressed deltagamma1 after being irradiated in vivo. p53 mRNA levels in irradiated wild-type and deltagamma 1-expressing cells were both only slightly elevated, suggesting that Mdm2 regulates p53 levels by a post-transcriptional mechanism. p53-mediated induction of the pro-apoptotic gene encoding Bax was also significantly lower in F10deltagamma1 cells irradiated in vivo. Moreover, F10deltagamma1 and BL6 cells were less apoptotic than F10 cells when the cells were irradiated in vivo. The p53 in F10 cells appears to be as functional as that in NIH3T3 cells because irradiation-induced expression of p53-target genes was comparable in both cells. Collectively, deltagamma1 appears to reduce irradiation-induced Mdm2 phosphorylation, which then blocks irradiation-stimulated p53 accumulation. Defects, such as deltagamma1, in PP2A may thus contribute to melanoma cell radioresistance.
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PMID:A truncated isoform of the PP2A B56gamma regulatory subunit reduces irradiation-induced Mdm2 phosphorylation and could contribute to metastatic melanoma cell radioresistance. 1502

Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha. Metabolic labeling with (32)P-orthophosphate in the transfected cells revealed that there is no major phosphorylation site other than the three residues in PKBalpha. PKBalpha activated by heat shock was more stable than the enzyme stimulated by PDGF in the cells, and PKBalpha recovered from heat-shocked cells was resistant to the protein phosphatase treatment, whereas the enzyme obtained from the growth-factor-stimulated cells was inactivated by dephosphorylation. Heat shock also enhanced the association of the PH-domain fragment to the full-length PKBalpha in the transfected cells. On the other hand, the PH-domain fragment of PKBalpha, which moves from the cytosol to the plasma membrane upon PDGF stimulation by the interaction with the phosphatidylinositol 3-kinase products, did not translocate but stayed in the cytosol in heat-shocked NIH 3T3 cells. Furthermore, PKBalpha was associated with the nuclear region in heat-shocked cells, which is not observed in growth-factor-stimulated cells. These results indicate that heat shock induces the conformational change of PKBalpha that accompanies the protein complex formation and perinuculear/nuclear localization of the enzyme, to generate an active form by a mechanism distinct from that in the growth-factor-signaling pathway.
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PMID:Distinct activation mechanisms of protein kinase B by growth-factor stimulation and heat-shock treatment. 1506 72


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