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 addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.
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PMID:Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity. 132 19

Certain waterblooms of toxic cyanobacteria (blue-green algae) are a health threat because of their production of toxic peptides, termed microcystins, which cause liver damage in wild and domesticated animals. The most widely studied microcystin is microcystin-LR, a heptapeptide containing the two L-amino acids, leucine and arginine. The inhibition of protein phosphatase type 1 and type 2A activities by microcystin-LR is similar to that of the known protein phosphatase inhibitor and tumor promoter okadaic acid. We show in this report that microcystin-LR, applied below the acute toxicity level, dose-dependently increases the number and percentage area of positive foci for the placental form of glutathione S-transferase in rat liver, which was initiated with diethylnitrosamine. The result was obtained independently through two animal experiments. This observation indicates that microcystin-LR is a new liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. This provides further evidence that the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver.
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PMID:Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. 161 89

A gene named stp1+, coding for a 17.5-kDa protein, that rescues cdc25-22 when overexpressed, has been previously isolated from fission yeast. Here we describe the expression and purification of Stp1 protein as a fusion with the glutathione S-transferase in E. coli and its kinetic characterisation. Stp1 deduced protein sequence shows an high homology to members of a class of cytosolic low M(r) protein phosphatase previously known to exist only in mammalian species. Stp1 has a kinetic behaviour that appears to be intermediate with respect to the two isoenzymatic forms of low M(r) protein tyrosine phosphatases present in mammalian tissues. These differing kinetic characteristics are mainly due to the sequence 45-56 that is spatially close to the active site pocket.
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PMID:Expression, purification and kinetic behaviour of fission yeast low M(r) protein-tyrosine phosphatase. 749 7

p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
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PMID:Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1. 768 43

We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase. 772 17

The expression of the Epstein-Barr virus LMP1 oncogene is regulated by viral and non-viral factors in a tissue dependent fashion. The virus encoded transcription factor EBNA2 induces its expression in human B-cells. However, this induction also requires the contribution of cellular and/or other viral factors. In nasopharyngeal carcinoma cells and in cells from Hodgkin's lymphoma, LMP1 gene transcription is independent of viral products. Here we show that the effect of a factor binding to a cAMP responsive-like element (CRE) in the LMP1 gene transcription regulatory sequence (LRS) is essential for efficient promoter activity in the DG75 B-cell line and that elevation of cAMP levels in the cells induces LRS-derived CAT activity in a CRE dependent fashion. Incubation of two EBV-immortalized B-cell lines expressing endogenous EBNA2A with 8-Br cAMP increased the levels of the latency associated 66 kDa LMP1 within 2 h. Interestingly, LMP1 expression in DG75 cells conferred resistance to the inhibitory effect of 8-Br cAMP on cell proliferation. The protein phosphatase 1 and 2A (PP1 and PP2A, respectively) inhibitor okadaic acid also stimulated LRS-CAT activity in DG75 cells. EBNA2A from an EBV-immortalized B-cell line co-immunopurified with a PP1-like protein. An EBNA2A fragment spanning residues 324-436 fused to the GST protein specifically rescued a PP1/PP2A-like component from DG75 cell extracts. This GST-EBNA2A fusion product inhibited a PP1-like activity in nuclear extracts from these cells.
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PMID:Response to cAMP levels of the Epstein-Barr virus EBNA2-inducible LMP1 oncogene and EBNA2 inhibition of a PP1-like activity. 781 42

Sequence analysis of the genomes of the Leporipoxviruses myxoma virus and Shope fibroma virus (SFV) led to the discovery of open reading frames homologous to the vaccinia H1L gene encoding a soluble protein phosphatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma BamHI-I fragment and the SFV BamHI-M fragment, and the resulting encoded proteins were designated I1L and M1L, respectively. The localization and orientation of the myxoma I1L and SFV M1L open reading frames within the well conserved central core of the viral genomes closely mirror that of the Orthopoxviruses vaccinia virus and variola virus. The myxoma I1L and SFV M1L phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cysteine, found previously to be essential for phosphotyrosine dephosphorylation. The vaccinia H1L phosphatase was originally shown to have the ability to dephosphorylate phosphotyrosyl and phosphoseryl residues in vitro. To assess whether this is a common feature of poxvirus phosphatases, myxoma I1L was expressed as a GST-fusion protein, purified, and shown to dephosphorylate substrates containing tyrosine and serine phosphorylated residues, in a similar fashion to vaccinia H1L. A myxoma I1L variant, in which the active site cysteine 110 was mutated to serine, was expressed in a parallel fashion to the wild-type I1L protein and found to be completely deficient in its ability to dephosphorylate both phosphotyrosine and phosphoserine amino acids. In an attempt to ascertain the biological requirement for the myxoma I1L phosphatase, we constructed a recombinant myxoma virus containing a disrupted I1L open reading frame. This I1L mutant virus was able to successfully propagate in tissue culture only in the presence of a wild-type complementing gene, and pure virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma I1L dual specificity phosphatase is an essential factor for virus viability.
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PMID:Myxoma virus and Shope fibroma virus encode dual-specificity tyrosine/serine phosphatases which are essential for virus viability. 783 13

The major protein kinase activity from vaccinia virus core particles was purified to near homogeneity. The protein kinase is a 50-kDa polypeptide that is shown here to phosphorylate primarily seryl residues in alpha-casein, a casein kinase I-specific peptide substrate, and itself through autophosphorylation. The sequence of four peptides derived from the protein kinase demonstrated that it is encoded by the vaccinia virus F10L gene. Expression of the F10L gene product in bacteria as a fusion with glutathione S-transferase confirmed that the vaccinia F10L gene encodes the protein kinase. We have termed this enzyme vaccinia protein kinase 2 (VPK2) to distinguish it from the protein kinase encoded by the vaccinia B1R gene. Targeted disruption of the VPK2 gene with a positive selectable marker demonstrated that all viruses with a disrupted gene also possessed a wild-type gene, suggesting that VPK2 is essential for viability. The discovery of a second essential protein kinase encoded by vaccinia virus, in addition to a protein phosphatase, underscores the importance of protein phosphorylation in poxvirus biogenesis.
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PMID:Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus. 805 37

Specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-ribosylation.
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PMID:ADP-ribosylation of Rho proteins by Clostridium botulinum exoenzyme C3 is influenced by phosphorylation of Rho-associated factors. 819 24

We have characterized a growth factor-inducible gene, erp, and demonstrated that it encodes a 367-amino-acid nontransmembrane tyrosine phosphatase protein with significant similarity to the vaccinia virus H1 protein. Immunoprecipitation analyses show that the erp protein, ERP, is rapidly induced following serum stimulation of quiescent fibroblasts. ERP has been expressed as a fusion protein with glutathione S-transferase and shown to have tyrosine as well as serine protein phosphatase activity. The enzymatic activity of ERP depends on the presence of reducing agents such as dithiothreitol, and its tyrosine phosphatase activity is inhibited by sodium vanadate, a potent inhibitor of protein tyrosine phosphatases. The number of stable NIH 3T3 clones obtained after transfection with a vector expressing the complete ERP protein is reduced more than 90% compared with that after transfection with a vector expressing a mutated inactive ERP protein. The remaining ERP-expressing clones present a significant increase in the proportion of bi- and multinucleated cells and a decrease in proliferation rate. Studies on the genomic structure reveal that the erp transcription unit is 2.8 kbp long and split into four exons. The erp gene maps to the 17A2-17C region of the murine genome. Our results demonstrate that the protein product of the immediate-early gene erp has a negative effect on cell proliferation.
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PMID:Structure, mapping, and expression of erp, a growth factor-inducible gene encoding a nontransmembrane protein tyrosine phosphatase, and effect of ERP on cell growth. 835 78


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