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)

Transient transfection of COS-1 cells with an expression vector for NIPP-1, a nuclear subunit of protein phosphatase-1, did not result in an overexpression of NIPP-1 protein, although the levels of mRNA encoding NIPP-1 increased dramatically. Moreover, high concentrations of NIPP-1 mRNA inhibited the translation in reticulocyte lysates of various unrelated mRNAs. This inhibition of translation was caused by the NIPP-1 messenger and not by the translation product, since mutation of the start codon abolished NIPP-1 protein production, but had no influence on the translational inhibition. Analysis of deletion mutants showed that the inhibition was mediated by a 0.5-kb fragment in the 5'-end of the NIPP-1 mRNA. This region, when inserted in the 5'-untranslated region of the beta-galactosidase messenger, inhibited the translation of beta-galactosidase mRNA in COS-1 cells. A predicted highly stable secondary structure deltaG = -239.5 kJ/mol) is present between residues 300 and 500 of NIPP-1 mRNA. The possible importance of this structure in the translational inhibition is discussed.
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PMID:Inhibition of translation by mRNA encoding NIPP-1, a nuclear inhibitor of protein phosphatase-1. 924 54

Specific rabbit polyclonal antibodies against peptides corresponding to the highly homologous protein serine/threonine phosphatase 2A and X catalytic subunits (PP2A/C and PPX/C respectively) were used to investigate the cellular and subcellular distribution of PP2A/C and PPX/C, as well as their methylation state. Immunoblots of rat tissue extracts revealed a widespread distribution of these enzymes but particularly high levels of PP2A/C and PPX/C in brain and testes respectively. In addition, immunoblots of subcellular fractions and immunocytochemical analyses of rat brain sections demonstrated that PPX/C is predominantly localized to the nucleus, whereas PP2A/C is largely cytoplasmic. Treatment of nuclear extracts with alkali resulted in increased PPX/C immunoreactivity to a polyclonal antibody directed against the C-terminus; no change in PPX immunoreactivity was observed using an antibody against an internal peptide. Alkali treatment of brain and liver cytosolic and nuclear extracts did not change the molecular mass or the isoelectric point of PPX/C. Furthermore, tritiated PPX/C was immunoprecipitated from COS cell extracts incubated with the methyl donor S-adenosyl-l-[methyl-3H]methionine. Thus the increase in immunoreactivity probably results from removal of a carboxymethyl group from PPX/C, as has been shown previously for PP2A/C [Favre, Zolnierowicz, Turowski and Hemmings (1994) J. Biol. Chem. 269, 16311-16317]. Together, our results indicate that the PPX catalytic subunit is a predominantly nuclear phosphatase and is methylated at its C-terminus.
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PMID:Carboxymethylation of nuclear protein serine/threonine phosphatase X. 935 19

The transcription factor Elk-1 is a component of ternary complex factor and regulates gene expression in response to a wide variety of extracellular stimuli. Phosphorylation of the C-terminal domain of Elk-1, especially at serine 383, is important for its transactivation activity. Recently mitogen-activated protein kinases, such as extracellular signal-regulated kinase, stress-activated protein kinase, and p38 mitogen-activated protein kinase have been demonstrated to be Elk-1 kinases. However, negative regulators of Elk-1, such as protein phosphatases, still remain to be identified. Here we report that COS cell lysates were able to dephosphorylate an extracellular signal-regulated kinase-phosphorylated glutathione S-transferase-Elkc fusion protein, including serine 383. The phosphatase activity was inhibited by cyclosporin A (a calcineurin inhibitor) but not by okadaic acid (a PP1 and PP2A inhibitor). Purified calcineurin also could efficiently dephosphorylate glutathione S-transferase-Elkc in vitro. Pretreatment of COS cells with cyclosporin A significantly enhanced epidermal growth factor-induced serine 383 Elk-1 phosphorylation whereas ionomycin inhibited the Elk-1 phosphorylation. These data provide both in vitro and in vivo evidence that calcineurin is the major Elk-1 phosphatase and plays a critical role in Elk-1 regulation. The identification of calcineurin as the major Elk-1 phosphatase may provide a mechanism for Elk-1 regulation by Ca2+ signals as well as a possible biochemical basis for the neurotoxicity and nephrotoxicity of the immunosuppressant drug cyclosporin A.
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PMID:The calcium/calmodulin-dependent protein phosphatase calcineurin is the major Elk-1 phosphatase. 936 95

We demonstrated previously that PP2A exists in many cell types as two abundant forms: (1) holoenzyme composed of two regulatory subunits, A and B, and a catalytic subunit C; and (2) core enzyme consisting of the A and C subunits. These two forms have different substrate specificities. Since published data suggested that HIV-1 transcription may be regulated by a cellular protein phosphatase, it was of interest to determine whether changing the ratio between PP2A core and holoenzyme affects HIV-1 gene expression. This question was addressed by expression in COS cells of an N-terminal mutant of the A subunit, A delta 5, which binds the C but not the B subunit. This resulted in an increase in the amount of core enzyme and a decrease in the amount of holoenzyme concomitant with the expected change in phosphatase activity. Tat-stimulated transcription from the HIV-1 LTR was inhibited 5-fold by mutant A delta 5, whereas mRNA synthesis directed by the actin promoter was not affected. Furthermore, virus production in COS, HeLa, and Jurkat T cells was inhibited 45-, 5-, and 3-fold, respectively, by mutant A delta 5. These results demonstrate that the balance between PP2A holoenzyme and core enzyme is important for HIV-1 gene expression and virus production.
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PMID:Increasing the ratio of PP2A core enzyme to holoenzyme inhibits Tat-stimulated HIV-1 transcription and virus production. 940 Jun 15

The 75-kDa inositol polyphosphate 5-phosphatase (5-phosphatase II) hydrolyzes various signaling molecules including the following: inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4, 5-trisphosphate. Although studied extensively, a demonstrably full-length cDNA encoding 5-phosphatase II has yet to be isolated. In this study we used a human partial 2.3-kilobase pair (kb) cDNA to screen mouse brain and kidney cDNA libraries, resulting in the isolation of a 3.7-kb cDNA (M5), which by multiple criteria represents a full-length cDNA encoding a 115-kDa 5-phosphatase II. We also isolated a smaller cDNA (M22) with a unique N terminus that encodes a 104-kDa polypeptide. Analysis of these cDNAs suggests a further 87-kDa isoform may arise from differential splicing resulting in translation at methionine 234 in M5. RNA analysis of tissues demonstrates expression of two mRNA species of approximately 4.0 or 3.0 kb, respectively. Probes unique to the 5' end of M5 or M22 hybridized to the 4.0- or 3.0-kb transcripts, respectively. RNA analysis using probes derived from sequence 3' to the potential splice site in M5 and M22 hybridized to both transcripts. Expression of the recombinant 115-kDa protein, or a smaller recombinant protein lacking the N terminus transiently in COS-7 cells, showed localization of enzyme activity to the membrane. Removal of the C-terminal CAAX motif resulted in a significant translocation of the protein lacking the N terminus but not the 115-kDa 5-phosphatase to the cytosol. Western blot analysis of membrane and cytosolic fractions of multiple mouse tissues confirmed the 115-kDa 5-phosphatase II was located in the membrane, whereas the 104- and 87-kDa isoforms were prominent in the cytosol. Collectively these studies demonstrate the widespread expression of at least three isoforms of 5-phosphatase II derived from RNA splicing events. This allows differential distribution of the 5-phosphatase II activity between the membrane and cytosol of the cell and thereby may regulate enzyme access to phosphoinositide-derived signaling molecules.
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PMID:Distinct membrane and cytosolic forms of inositol polyphosphate 5-phosphatase II. Efficient membrane localization requires two discrete domains. 952 32

Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. However, mechanisms responsible for ceramide-induced apoptosis remain unclear. We investigated the possibility that ceramide may decrease antiapoptotic signaling in cells by inhibiting Akt kinase activity. Our data show that C2-ceramide induces apoptosis in HMN1 motor neuron cells and decreases both basal and insulin- or serum-stimulated Akt kinase activity 65-70%. These results are consistent with decreased Akt kinase activity being involved in the apoptotic effects of ceramide. This possibility is further supported by studies showing that constitutively active Akt kinase decreases C2-ceramide-induced death of HMN1 cells as well as COS-7 cells. Decreased Akt activity is not due to ceramide activating the ceramide-activated protein phosphatase or to a direct inhibition of Akt kinase by ceramide, suggesting that ceramide acts upstream of Akt kinase to decrease its activity. Treating cells with C2-ceramide does not affect phosphorylation of insulin receptor substrate-1, interactions between insulin receptor substrate-1 and p85, or insulin-stimulated phosphatidylinositol 3-kinase activity, suggesting that the effects of C2-ceramide on Akt kinase are not mediated through modulating phosphatidylinositol 3-kinase. In sum, our results suggest that inhibition of the key antiapoptotic kinase, Akt, may play an important role in ceramide-induced apoptosis.
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PMID:Inhibition of Akt kinase by cell-permeable ceramide and its implications for ceramide-induced apoptosis. 963 28

The rotavirus nonstructural phosphoprotein NSP5 is encoded by a gene in RNA segment 11. Immunofluorescence analysis of fixed cells showed that NSP5 polypeptides remained confined to viroplasms even at a late stage when provirions migrated from these structures. When NSP5 was expressed in COS-7 cells in the absence of other viral proteins, it was uniformly distributed in the cytoplasm. Under these conditions, the 26-kDa polypeptide predominated. In the presence of the protein phosphatase inhibitor okadaic acid, the highly phosphorylated 28- and 32- to 35-kDa polypeptides were formed. Also, the fully phosphorylated protein had a homogeneous cytoplasmic distribution in transfected cells. In rotavirus SA11-infected cells, NSP5 synthesis was detectable at 2 h postinfection. However, the newly formed 26-kDa NSP5 was not converted to the 28- to 35-kDa forms until approximately 2 h later. Also, the protein kinase activity of isolated NSP5 was not detectable until the 28- and 30- to 35-kDa NSP5 forms had been formed. NSP5 immunoprecipitated from extracts of transfected COS-7 cells was active in autophosphorylation in vitro, demonstrating that other viral proteins were not required for this function. Treatment of NSP5-expressing cells with staurosporine, a broad-range protein kinase inhibitor, had only a limited negative effect on the phosphorylation of the viral polypeptide. Staurosporine did not inhibit autophosphorylation of NSP5 in vitro. Together, the data support the idea that NSP5 has an autophosphorylation activity that is positively regulated by addition of phosphate residues at some positions.
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PMID:Analysis of rotavirus nonstructural protein NSP5 phosphorylation. 965 80

The Tpl-2 kinase activates the nuclear factor of activated T cells (NFAT) and induces IL-2 expression in T-cell lines. Here we show that the activation of the IL-2 promoter by Tpl-2 is inhibited by mutant signaling molecules that inhibit the mitogen-activated protein kinase (MAPK) or the calcineurin/NFAT pathways and is promoted by combinations of signaling molecules that activate these pathways. We, therefore, conclude that signals generated by the convergence of the MAPK and the calcineurin/NFAT pathway are necessary and sufficient for the activation of the IL-2 promoter by Tpl-2. The activation of both the IL-2 promoter and an NFAT-driven minimal promoter were shown to depend on signals transduced by Raf1. However, it was only the IL-2 promoter whose activation by Tpl-2 was fully blocked by the dominant negative mutant MEK1S218/222A and the MEK1/MEK2 inhibitor PD098059. Since the activation of NFAT is MAPK-dependent these findings suggested that the activation of MAPK by Tpl-2 is either independent or only partially dependent on MEK1 and MEK2. In addition, they suggested that the activation of the IL-2 promoter is under the control of not only NFAT but also a second factor whose activation is MEK-dependent. Experiments in COS-1 and EL-4 cells confirmed both hypotheses and revealed that the second factor activated by Tpl-2 is NF-kappaB. While the activation of the IL-2 promoter and an NFAT-driven minimal promoter by Tpl-2 was fully blocked by the dominant negative mutant NFAT delta418, it was only partially blocked by the calcineurin inhibitor cyclosporin A suggesting that the Tpl-2-mediated NFAT activation is under the control of a combination of calcineurin-dependent and independent pathways. Both pathways were fully blocked by Bcl-2 or Bcl-X(L).
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PMID:Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB. 984 Sep 24

Methylation of the C-terminal leucine residue (Leu309) of protein serine/threonine phosphatase 2A catalytic subunit (PP2AC) is known to regulate catalytic activity in vitro, but the functional consequence(s) of this post-translational modification in the context of the cell remain unclear. Alkali-induced demethylation of PP2AC in purified PP2A heterotrimer (ABalphaC), but not in purified PP2A heterodimer (AC), indicated that a larger fraction of PP2AC is carboxymethylated in ABalphaC than in AC. To explore the role of Leu309 in PP2A holoenzyme assembly, epitope-tagged PP2A catalytic subunit (HA-PP2A) and a mutant of HA-PP2A containing an alanine residue in place of Leu309 (HA-PP2A-L309A) were transiently expressed in COS cells. Both recombinant proteins exhibited serine/threonine phosphatase activity when immunoisolated from COS cell extracts. HA-PP2A, but not HA-PP2A-L309A, was carboxymethylated in vitro. A chromatographic analysis of cell extracts indicated that most endogenous PP2AC and HA-PP2A were co-eluted with the A and Balpha regulatory subunits of PP2A, whereas most HA-PP2A-L309A seemed to elute with the A subunit as a smaller complex or, alternatively, as free catalytic (C) subunit. The A subunit co-immunoisolated with both tagged proteins; however, substantially less Balpha subunit co-immunoisolated with HA-PP2A-L309A than with HA-PP2A. These results demonstrate that the reversibly methylated C-terminal leucine residue of PP2AC is important for Balpha regulatory subunit binding. Furthermore, the results provide evidence for an interrelationship between PP2AC carboxymethylation and PP2A holoenzyme assembly.
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PMID:Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Balpha subunit. 1019 Dec 53

Axin forms a complex with glycogen synthase kinase-3beta (GSK-3beta) and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin, thereby stimulating the degradation of beta-catenin. Because GSK-3beta also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3beta inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3beta were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3beta, inhibited the phosphorylation of Axin by GSK-3beta in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated beta-catenin in L cells and expression of Dvl-1(DeltaPDZ), in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl.
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PMID:Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability. 1019 36


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