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)

Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.
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PMID:The protein phosphatase calcineurin determines basal parathyroid hormone gene expression. 1551 34

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of lphaand gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.
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PMID:Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells. 1555 48

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post-translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK-293 cells as a His-tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP lambda) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.
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PMID:New evidences for a regulation of deoxycytidine kinase activity by reversible phosphorylation. 1557 Dec 59

In this study, we show that protein phosphatase-1 (PP1) inhibitor-3 (Inh3) is localized to the nucleoli and centrosomes in interphase HEK 293 cells. Inh3 exhibited a specific co-localization to the nucleoli with PP1gamma1, and to the centrosomes with PP1alpha. These findings indicate that Inh3 may act as a modulator of PP1 functions in the processes of cytokinesis, as well as of nucleolar events. The specificity of the interaction of Inh3 with the PP1 isoforms was also demonstrated in vitro, where Inh3 co-immunoprecipitated with PP1alpha and PP1gamma1, but not with PP1beta. The nuclear localization signal of Inh3 was identified as a N-terminal basic cluster (33RKRK36), while nucleolar localization was shown to be dependent on a C-terminal basic cluster (94HRKGRRR100). The importance of the individual basic residues was quantitatively assessed by site-directed mutagenesis and a novel use of laser scanning cytometry.
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PMID:Protein phosphatase-1 inhibitor-3 is co-localized to the nucleoli and centrosomes with PP1gamma1 and PP1alpha, respectively. 1625 67

ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15-20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.
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PMID:Characterization of the reversible phosphorylation and activation of ERK8. 1633 13

The circadian clock is regulated by a transcription/translation negative feedback loop. A key negative regulator of circadian rhythm in mammals is the PER2 (mammalian PERIOD 2) protein. Its daily degradation at the end of the night accompanies de-repression of transcription. CKI (casein kinase I ) has been identified as the kinase that phosphorylates PER2, targeting it for ubiquitin-mediated proteasomal degradation. We now report that PER2 degradation is also negatively regulated by PP1 (protein phosphatase 1)-mediated dephosphorylation. In Xenopus egg extract, PP1 inhibition by Inhibitor-2 accelerated mPER2 degradation. Co-immunoprecipitation experiments showed that PER2 bound to PP1c in transfected HEK-293 cells. PP1 immunoprecipitated from HEK-293 cells, mouse liver and mouse brain, dephosphorylated CKI-phosphorylated PER2, showing that PER2 is a substrate for mammalian endogenous PP1. Moreover, over-expression of the dominant negative form of PP1c, the D95N mutant, accelerated ubiquitin and proteasome-mediated degradation of PER2, and shortened the PER2 half-life in HEK-293 cells. Over-expression of the PP1 inhibitors, protein phosphatase 1 holoenzyme inhibitor-1 and Inhibitor-2, confirmed these results. Thus PP1 regulates PER2 stability and is therefore a candidate to regulate mammalian circadian rhythms.
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PMID:Protein phosphatase 1 regulates the stability of the circadian protein PER2. 1695 25

Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.
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PMID:Inhibition of protein phosphatase 2A activity by selective electrophile alkylation damage. 1690 60

The transcription factor Egr-1 activates cyclin-dependent protein kinase 5 (Cdk5) during nerve growth factor (NGF)-induced differentiation of PC12 cells into neurons (Harada, T. Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459). The downstream target of Cdk5 in the Egr-1/Cdk5 pathway is not clear. In this study, we observed that phosphorylation of protein phosphatase 1 (PP1) on Thr(320) is reduced in brain extracts from Egr-1(-/-) mice, indicating that a kinase downstream of Egr-1 phosphorylates PP1. In HEK 293 cells co-transfected with PP1 and Cdk5, Cdk5 phosphorylates PP1. In vitro, Cdk5 purified from bovine brain phosphorylates bacterially expressed recombinant PP1. In NGF-treated PC12 cells, inhibition of Cdk5 by olomoucine or silencing Cdk5 expression by small interfering RNA strategy, suppresses PP1 phosphorylation. Silencing Cdk5 expression by small interfering RNA also blocks NGF-induced neurite outgrowth. Overexpression of PP1 (wild type) promotes NGF-induced differentiation of PC12 cells, whereas that of PP1 (T320A) has no effect. Our data indicate that PP1 is a downstream target of the NGF/Egr-1/Cdk5 pathway during NGF-induced differentiation of PC12 cells and suggest that PP1 phosphorylation promotes neuronal differentiation.
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PMID:Phosphorylation of protein phosphatase 1 by cyclin-dependent protein kinase 5 during nerve growth factor-induced PC12 cell differentiation. 1720 32

The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr(888) contributing significantly to this effect. To determine whether CaR(T888) is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaR(pT888)). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaR(T888A), phorbol ester (PMA) treatment increased CaR(pT888) immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca(2+) concentration from 0.5 to 2.5 mM increased CaR(T888) phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca(2+) and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaR(T888A) did exhibit increased apparent agonist sensitivity, by converting intracellular Ca(2+) (Ca(2+)(i)) oscillations to sustained plateau responses in some cells, we still observed Ca(2+)(i) oscillations in a significant number of cells. This suggests that CaR(T888) contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca(2+)(i) oscillations. Finally, dephosphorylation of CaR(T888) was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca(2+)(i) oscillations. In addition, calyculin/PMA cotreatment increased CaR(T888) phosphorylation in bovine parathyroid cells. Therefore, CaR(T888) is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.
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PMID:Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity. 1737 81

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.
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PMID:Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon. 1745 47


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