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)

Tyrosine phosphoproteins of size 115-120 kDa were purified from membranes of chicken embryo fibroblasts (CEF) infected with Rous sarcoma virus (RSV). A mouse was immunized with these proteins, and the immune serum was used to screen a CEF cDNA expression library. A highly immunoreactive clone (KS5) was identified and characterized. The cDNA of this clone is 2.3 kb in length with a short 5' UTR and a single major open reading frame (ORF) encoding a polypeptide of 719 amino acids, with a calculated molecular weight of 81.1 kDa. The encoded protein contains an amino terminal PDZ domain, followed by a predicted coiled-coil region, a PEST domain, and a carboxy-terminal SAM domain. Consensus sequence motifs for tyrosine phosphorylation are also present, as are consensus sequences for the binding of SH2 and PDZ domains. Antisera from mice immunized with bacterially expressed fragments of the KS5 protein recognized proteins of size 230, 116, and 65 kDa in CEF. In other chicken embryo tissues, a 116-kDa species was the predominant protein recognized. The 116-kDa species is tyrosine-phosphorylated in RSV-CEF. The presence of PDZ and SAM domains in the KS5 protein suggests that it may act as a molecular adaptor, promoting and relaying information in a signal transduction pathway. It is a member of a family of related proteins, all of which have a highly conserved PDZ domain adjacent to a coiled-coil region. Two other members of this family are the neuronal proteins spinophilin (Allen, P.B., Ouimet, C.C., Greengard, P., 1997. Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines. Proc. Natl. Acad. Sci. USA 94, 9956-9961) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A. , Takai, Y., 1997. Neurabin: A novel neural tissue-specific actin filament-binding protein involved in neurite formation. J. Cell Biol. 139, 951-961).
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PMID:An avian cDNA encoding a tyrosine-phosphorylated protein with PDZ, coiled-coil, and SAM domains. 975 12

Tau hyperphosphorylation is a central event in the development of Alzheimer's Disease (AD). Protein phosphatase 2A (PP2A) heterotrimer formation is necessary for efficient dephosphorylation of the tau protein. S-Adenosylmethionine-dependent carboxyl methylation is essential for the assembly of PP2A heterotrimers. Epidemiological evidence indicates that elevated plasma homocysteine is an independent risk factor for AD. Homocysteine is a key intermediate in the methyl cycle and elevated plasma homocysteine results in a global decrease in cellular methylation. We propose that the PP2A methylation system is the link relating elevated plasma homocysteine to AD.
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PMID:Protein phosphatase 2A methylation: a link between elevated plasma homocysteine and Alzheimer's Disease. 1199 7

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.
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PMID:Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes. 1269 24

14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3-Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-D-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.
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PMID:14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking. 1506 4

S-adenosylmethionine (SAMe) and its metabolite 5'-methylthioadenosine (MTA) are proapoptotic in HepG2 cells. In microarray studies, we found SAMe treatment induced Bcl-x expression. Bcl-x is alternatively spliced to produce two distinct mRNAs and proteins, Bcl-x(L) and Bcl-x(S). Bcl-x(L) is antiapoptotic, while Bcl-x(S) is proapoptotic. In this study we showed that SAMe and MTA selectively induced Bcl-x(S) in a time- and dose-dependent manner in HepG2 cells. There are three transcription start sites in the human Bcl-x gene which yield only Bcl-x(L) in control HepG2 cells. SAMe and MTA treatment did not affect promoter usage, but while one promoter yielded only Bcl-x(L), the other two yielded both Bcl-x(L) and Bcl-x(S), with Bcl-x(S) as the predominant messenger RNA (mRNA) species. Trichostatin A, 3-deaza-adenosine, cycloleucine, and okadaic acid had no effect on Bcl-x(S) induction by SAMe or MTA. Calyculin A and tautomycin, on the other hand, blocked SAMe and MTA-mediated Bcl-x(S) induction and apoptosis in a dose-dependent manner. SAMe and MTA increased protein phosphatase 1 (PP1) catalytic subunit mRNA and protein levels and dephosphorylation of serine-arginine proteins, with the latter blocked by calyculin A. The effects of SAMe and MTA on Bcl-x(S), PP1 expression, and apoptosis were also seen in 293 cells, but not in primary hepatocytes. Induction of Bcl-x(S) by ceramide in HepG2 cells also resulted in apoptosis. In conclusion, we have uncovered a highly novel action of SAMe and MTA, namely the ability to affect the cellular phosphorylation state and alternative splicing of genes, in this case resulting in the induction of Bcl-x(S) leading to apoptosis.
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PMID:S-adenosylmethionine and its metabolite induce apoptosis in HepG2 cells: Role of protein phosphatase 1 and Bcl-x(S). 1523 6

It has been reported that S-adenosylmethionine-dependent protein methylation in rat kidney extracts can be greatly stimulated by tyrphostin A25, a tyrosine kinase inhibitor. We have investigated the nature of this stimulation. We find that addition of tyrphostin A25, in combination with the protein phosphatase inhibitor vanadate, leads to the stimulation of methylation of polypeptides of 64, 42, 40, 36, 31, and 15 kDa in cytosolic extracts of mouse kidney. The effect of tyrphostin appears to be relatively specific for the A25 species. The enhanced methylation does not represent the activity of the families of protein histidine, lysine or arginine methyltransferases, nor that of the l-isoaspartyl/d-aspartyl methyltransferase, enzymes responsible for the bulk of protein methylation in most cell types. Chemical and enzymatic analyses of the methylated polypeptides suggest that the methyl group is in an ester linkage to the protein. In heart extracts, we find a similar situation but here the stimulation of methylation is not dependent upon vanadate and an additional 18 kDa methylated species is found. In contrast, little or no stimulation of methylation is found in brain or testis extracts. This work provides evidence for a novel type of protein carboxyl methylation reaction that may play a role in signaling reactions in certain mammalian tissues.
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PMID:A new type of protein methylation activated by tyrphostin A25 and vanadate. 1552 82

Emerging evidence suggests critical roles for protein phosphatase 2A (PP2A) in islet beta cell function, including survival and demise (Kowluru A: Biochemical Pharmacol 69:1681-1691, 2005). Herein, we identified an okadaic acid (OKA)-sensitive PP2A-like phosphatase in the nuclear fraction from insulin-secreting INS-1 cells. Western blot analysis indicated relatively higher abundance of the catalytic subunit of protein phosphatase 4 (PP4c) compared to PP2Ac in this fraction. Autoradiographic and vapor-phase equilibration analyses suggested that the nuclear PP4c undergoes OKA-sensitive carboxylmethylation (CML) when S-adenosyl-L-((3)H-methyl) methionine (SAM) was used as the methyl donor. Exposure of INS cells to interleukin-1beta (IL-1beta; 600 pM; 48 h) resulted in a marked increase in nitric oxide (NO) release with concomitant reduction in the degree of expression, the CML and the catalytic activity of only PP4, but not PP2A, in the nuclear fraction. Immunoprecipitation studies suggested potential complexation of PP4c with nuclear lamin-B, a key regulatory protein involved in the nuclear envelope assembly. Based on these findings, we propose that IL-1beta-mediated inhibition of PP4 activity might result in the retention of lamin-B in its phosphorylated state, which is a requisite for its degradation by caspases leading to the apoptotic demise of the beta cell (Veluthakal et al.: Am J Physiol Cell Physiol 287:C1152-C1162, 2004).
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PMID:Localization of a nuclear serine/threonine protein phosphatase in insulin-secreting INS-1 cells: potential regulation by IL-1beta. 1683 Feb 32

Increased organ ischemia time leads to delayed graft function (DGF), increased acute rejection (AR), enhanced chronic allograft nephropathy (CAN), and reduced long-term allograft survival. The mechanisms by which IRI predisposes to AR and CAN are unknown. We hypothesized that gene expression profiling of ischemia-reperfusion injury (IRI)-affected kidney would identify how IRI predisposes to AR and CAN. Furthermore, we examined how current immunosuppressive drug molecular targets are altered by IRI. C57BL/6J mice were exposed to 30 (n = 3) or 60 (n = 3) minutes of bilateral kidney ischemia or sham surgery (n = 5). At 36 hour kidney tissue was collected and analyzed using Affymetrix 430MOEA (22626 genes) array and GC-RMA-SAM pipeline. Genes with the false discovery rate (q < 1%) and +/-50% fold change (FC) were considered affected by IRI. Genes coding for histocompatibility and antigen-presenting factors, calcineurin, and mammalian target of rapamycin (mTOR) pathway-associated proteins were selected using Gene Ontology (GO) analysis. GO analysis identified 10 and 17 alloimmunity-related genes affected by IRI induced by 30 and 60 minutes of ischemia, respectively, including Traf6 (FC = 2.99) and H2-D1 (FC = 2.58). We also detected significant IRI genomic responses in calcineurin and mTOR pathways represented by Fkbp5 (FC = 4.18) and Fkbp1a (FC = 2.0), and Eif4ebp1 (FC = 16.8) and Akt1 (FC = 3.64), respectively. These data demonstrated that IRI up-regulates expression of several alloimmunity-associated genes, which can in turn enhance alloimune responses. Our discovery of IRI-induced up-regulation of genes associated with calcineurin and mTOR pathways are consistent with clinical observations that FK506 and Rapamycin can alter the course of DGF. Further validation and dissection of these pathways can lead to novel approaches by which improved management of early "nonimmune" transplant events can decrease susceptibility to more classic "immune" changes and CAN.
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PMID:Genomic profiling of kidney ischemia-reperfusion reveals expression of specific alloimmunity-associated genes: Linking "immune" and "nonimmune" injury events. 1717 65

Alzheimer's disease (AD) neuropathology is characterized by the accumulation of phosphorylated tau and amyloid-beta peptides derived from the amyloid precursor protein (APP). Elevated blood levels of homocysteine are a significant risk factor for many age-related diseases, including AD. Impaired homocysteine metabolism favors the formation of S-adenosylhomocysteine, leading to inhibition of methyltransferase-dependent reactions. Here, we show that incubation of neuroblastoma cells with S-adenosylhomocysteine results in reduced methylation of protein phosphatase 2A (PP2A), a major brain Ser/Thr phosphatase, most likely by inhibiting PP2A methyltransferase (PPMT). PP2A methylation levels are also decreased after ectopic expression of PP2A methylesterase in Neuro-2a (N2a) cells. Reduced PP2A methylation promotes the downregulation of B alpha-containing holoenzymes, thereby affecting PP2A substrate specificity. It is associated with the accumulation of both phosphorylated tau and APP isoforms and increased secretion of beta-secretase-cleaved APP fragments and amyloid-beta peptides. Conversely, incubation of N2a cells with S-adenosylmethionine and expression of PPMT enhance PP2A methylation. This leads to the accumulation of dephosphorylated tau and APP species and increased secretion of neuroprotective alpha-secretase-cleaved APP fragments. Remarkably, hyperhomocysteinemia induced in wild-type and cystathionine-beta-synthase +/- mice by feeding a high-methionine, low-folate diet is associated with increased brain S-adenosylhomocysteine levels, PPMT downregulation, reduced PP2A methylation levels, and tau and APP phosphorylation. We reported previously that downregulation of neuronal PPMT and PP2A methylation occur in affected brain regions from AD patients. The link between homocysteine, PPMT, PP2A methylation, and key CNS proteins involved in AD pathogenesis provides new mechanistic insights into this disorder.
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PMID:Protein phosphatase 2A methyltransferase links homocysteine metabolism with tau and amyloid precursor protein regulation. 1736 Aug 97

Vitamin B12 (cobalamin, Cbl) is indispensable for proper brain development and functioning, suggesting that it has neurotrophic effects beside its well-known importance in metabolism. The molecular basis of these effects remains hypothetical, one of the reasons being that no efficient cell model has been made available for investigating the consequences of B12 cellular deficiency in neuronal cells. Here, we designed an approach by stable transfection of NIE115 neuroblastoma cells to impose the anchorage of a chimeric B12-binding protein, transcobalamin-oleosin (TO) to the intracellular membrane. This model produced an intracellular sequestration of B12 evidenced by decreased methyl-Cbl and S-adenosylmethionine and increased homocysteine and methylmalonic acid concentrations. B12 deficiency affected the proliferation of NIE115 cells through an overall increase in catalytic protein phosphatase 2A (PP2A), despite its demethylation. It promoted cellular differentiation by improving initial outgrowth of neurites and, at the molecular level, by augmenting the levels of proNGF and p75(NTR). The up-regulation of PP2A and pro-nerve growth factor (NGF) triggered changes in ERK1/2 and Akt, two signaling pathways that influence the balance between proliferation and neurite outgrowth. Compared with control cells, a 2-fold increase of p75(NTR)-regulated intramembraneous proteolysis (RIP) was observed in proliferating TO cells (P < 0.0001) that was associated with an increased expression of two tumor necrosis factor (TNF)-alpha converting enzyme (TACE) secretase enzymes, Adam 10 and Adam 17. In conclusion, our data show that B12 cellular deficiency produces a slower proliferation and a speedier differentiation of neuroblastoma cells through interacting signaling pathways that are related with increased expression of PP2A, proNGF, and TACE.
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PMID:Vitamin B12 deficiency reduces proliferation and promotes differentiation of neuroblastoma cells and up-regulates PP2A, proNGF, and TACE. 1995 61


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