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)

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS/PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of rat CNS (Walaas et al., 1983c). This acidic phosphoprotein has now been identified in bovine caudate nucleus cytosol and purified to homogeneity from this source. The purification procedure involved diethylaminoethyl-cellulose chromatography, ammonium sulfate fractionation, phenyl-Sepharose CL-4B chromatography, and fast protein liquid chromatography using Mono Q anion-exchange resin. Two isoforms of ARPP-21 (ARPP-21A and ARPP-21B) were obtained, which were present in approximately equal amounts in the starting material. ARPP-21A was purified 2610-fold with a final yield of 20% and ARPP-21B was purified 2940-fold with a final yield of 21%. The purified preparations of both isoforms were judged to be homogenous by SDS/PAGE. ARPP-21A and ARPP-21B yielded identical 2-dimensional thin-layer tryptic phosphopeptide maps, identical amino acid compositions and closely related, but distinct, reverse-phase high-pressure liquid chromatograms of tryptic digests. The amino acid composition of ARPP-21 showed a high content of glutamic acid/glutamine, and no methionine, tryptophan, tyrosine, phenylalanine, or histidine. ARPP-21 was stable to heat denaturation and to 50% (vol/vol) ethanol treatment and was partially soluble at pH 2. The Mr determined for ARPP-21 by SDS/PAGE was 21,000. The Stokes radius of ARPP-21 was 26.3 A, and the sedimentation coefficient of ARPP-21 was 1.3 S; these values yield a calculated molecular mass of 13,700 Da and a frictional ratio of 1.7, indicative of an elongated tertiary structure. ARPP-21 was an excellent substrate for cAMP-dependent protein kinase and was either not phosphorylated or only poorly phosphorylated by cGMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, calcium/calmodulin-dependent protein kinase II, casein kinase II, or protein kinase C. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 1.2 mol phosphate/mol purified ARPP-21. Phosphorylation occurred exclusively on seryl residues. Phospho-ARPP-21 was dephosphorylated effectively by protein phosphatase-1 or -2A, but not by protein phosphatase-2B or -2C. Rabbit polyclonal and mouse monoclonal antibodies were prepared to purified ARPP-21. These antibodies specifically immunoprecipitated ARPP-21, which was found to be highly enriched in the caudate nucleus and putamen of monkey brain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Purification and characterization of the protein from bovine caudate nucleus. 253 84

Calmodulin-dependent protein phosphatase (CaM-PPase) was isolated from bovine parotid gland by sequential application of DEAE-52, Affi-gel blue and calmodulin-affinity chromatography followed by gel filtration and high performance liquid chromatography. The enzyme was activated in the simultaneous presence of Ni2+ or Mn2+ and Ca2+ plus calmodulin. Ca2+/calmodulin-dependent activation of CaM-PPase was antagonized by inhibitors of calmodulin action, such as W-7 and trifluoperazine. Tryptophan fluorescence was quenched in the presence of Ni2+. CaM-PPase was a heterodimer. The molecular weights of large subunits which bound calmodulin (CaM) were 68 kD and 58 kD - the 68 kD subunit was predominant. Polyclonal antibodies against bovine calcineurin cross-reacted with both types of larger subunits. Using polyclonal antibodies against bovine calcineurin or the monoclonal antibody against subunit B of bovine calcineurin, the smaller molecular weight subunit (19 kD) was found to be immunologically identical to subunit B of bovine calcineurin. In bovine parotid gland, CaM-PPase was found both in acinar and duct cells.
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PMID:Ca2+/calmodulin-dependent protein phosphatase in bovine parotid gland: purification and characterization. 255 6

The complete amino acid sequence of bovine brain DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein, which is a potent and specific inhibitor of the catalytic subunit of protein phosphatase-1, has been determined. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage by endoproteinase Lys-C, endoproteinase Arg-C, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease, and to chemical cleavage by cyanogen bromide. The overlapping sets of peptides were purified by high performance liquid chromatography and subjected to amino acid sequencing by automated Edman degradation to deduce the complete sequence. The protein consists of a single NH2-terminal blocked polypeptide chain of 202 residues, with a calculated molecular mass of 22,591 daltons, excluding the unidentified NH2-terminal blocking group. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or hydrodynamic measurements. The threonine residue that is phosphorylated by cyclic AMP-dependent protein kinase (Hemmings, H. C., Jr., Williams, K. R., Konigsberg, W. H., and Greengard, P. (1984) J. Biol. Chem. 259, 14486-14490), and that must be phosphorylated for the expression of inhibitory activity, is located at position 34. The molecule contains only 1 cysteine residue and 1 tryptophan residue, at positions 72 and 161, respectively. DARPP-32 is very hydrophilic, and contains a stretch of 16 consecutive acidic residues from position 119 to 134. The predicted secondary structure suggests the presence of 47% alpha-helix, 7% beta-sheet, and 46% random coil, with 11 beta-turns. Comparison of the complete amino acid sequence of bovine DARPP-32 with that of rabbit skeletal muscle protein phosphatase inhibitor-1 revealed a significant amount of sequence identity in the NH2-terminal regions of these two proteins. The active region of inhibitor-1 has been localized to an NH2-terminal fragment (Aitken, A., and Cohen, P. (1982) FEBS Lett. 147, 54-58), the part of the molecule that is most similar to DARPP-32. These data suggest that these two protein phosphatase inhibitors may share a common structural basis for their inhibitory activity and may be related by a common ancestral gene.
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PMID:DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein. Primary structure and homology with protein phosphatase inhibitor-1. 351 Oct 54

It has been demonstrated earlier that the administration of tryptophan to fasted animals increased the levels of mRNA in the cytoplasm of the liver by stimulating the translocation of nuclear poly(A)-mRNA into the cytoplasm. Also, tryptophan increased the activity of hepatic nuclear envelope (NE) nucleoside triphosphatase, an enzyme considered to be involved in nucleocytoplasmic translocation of mRNA. In this study, the activities of two other NE-associated enzymes, protein phosphokinase and phosphoprotein phosphohydrolase, also implicated in nuclear RNA transport, were investigated in the livers of rats that received a single tube feeding of tryptophan. The administration of tryptophan to fasted rats 10 minutes before killing increased the hepatic NE activities of both enzymes, protein phosphokinase and phosphoprotein phosphohydrolase. Furthermore, tryptophan administration increased the in vivo incorporation of 3H-leucine into NE proteins (+83%) and into other subcellular fractions (+34 to +43%) of the liver compared with that into corresponding fractions of the control rats. Rats that received 3H-leucine to prelabel hepatic proteins and then were treated with puromycin to inhibit further protein synthesis followed by tube feeding of tryptophan revealed greater radioactivity associated with NE proteins than that in controls. These findings suggest that tryptophan may act to stimulate the transport or availability of proteins to the vicinity of the NE, possibly specific regulatory proteins, such as nucleoside triphosphatase, protein phosphokinase and phosphoprotein phosphohydrolase, which show an increase in activity and may then be responsible for the increase in the rate of nucleocytoplasmic translocation of mRNA.
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PMID:Effect of tryptophan on enzymes and proteins of hepatic nuclear envelopes of rats. 682 5

Polyomavirus middle T antigen (MT) interacts with several cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src (and the related kinases c-fyn and c-yes), and phosphatidylinositol-3 kinase. We made a single point mutation in MT, changing a conserved cysteine residue at position 120 to tryptophan, and characterized the biochemical and biological properties of the mutant (C120W) protein. The mutant MT protein does not associate with PP2A, pp60c-src, or phosphatidylinositol-3 kinase as judged by coimmunoprecipitation and associated phosphatase or kinase activity. The C120W mutant is defective in activation of c-fos expression and in morphological transformation of NIH 3T3 cells.
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PMID:Mutation of a cysteine residue in polyomavirus middle T antigen abolishes interactions with protein phosphatase 2A, pp60c-src, and phosphatidylinositol-3 kinase, activation of c-fos expression, and cellular transformation. 768 Mar 88

Fluorescence steady-state and lifetime measurements have been performed that permit the differentiation of the 2 intrinsic tryptophan residues in bovine low molecular weight phosphotyrosyl protein phosphatase (BPTP). Spectral information was obtained by use of two single-tryptophan mutant proteins, W39F and W49F, and the double mutant protein W39,49F. Fluorescence measurements show that Trp39 is characterized by a large blue shift, a low quantum yield, and a shorter mean lifetime compared to Trp49. Solute fluorescence quenching studies of W39F reveal that Trp49 is highly exposed to the aqueous environment. In contrast, Trp39 is situated within a hydrophobic core and is only partially accessible to quenching agents such as acrylamide, iodide ion, and cesium ion. The fluorescence contributions of Trp39 and Trp49 are additive, and their sum is equivalent to that observed for wild type BPTP. Calculated intramolecular distances between Trp39 or Trp49 and a 5-[[(acetylamino)-ethyl]amino]naphthalene-1- sulfonate group covalently bound at Cys12 or Cys17 of the respective protein mutants, place Trp49 within 10 A and Trp39 at least 20 A from the active site. The fluorescence decay of the single tryptophan mutants and, surprisingly, wild type BPTP were each adequately fitted as biexponentials. The latter is a consequence of the imprecision involved in determining actual minima in a three- and four-exponential fitting. Comparison of quenching results of wild type BPTP with those of the single tryptophan mutant proteins indicates that minor fluorescence components, easily resolved using a biexponential fitting for the mutant proteins, are unresolvable for wild type BPTP. These minor components skewed the weighted magnitudes and induced perturbations in lifetimes for the tryptophan fluorescence of wild type BPTP, which directly influenced the calculated values of Ksv and kq.
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PMID:Fluorescence resolution of the intrinsic tryptophan residues of bovine protein tyrosyl phosphatase. 787 23

We studied the effect of protein phosphatase and kinase inhibitors on Tax-mediated transcription of constructs carrying the reporter gene chloramphenicol acetyl transferase under the control of either the full-length LTR of HTLV-I or three copies of the tax-responsive 21-bp repeats. We observed that treatment with okadaic acid, which inhibits the serine/threonine protein phosphatases type 1 and 2A, reduced HTLV-I LTR transcriptional activation in MT2 and K562 cells; on the contrary, the enhancer activity of the 21-bp sequences was significantly increased in both cell lines; treatment with the protein kinase C inhibitor H-7 blocked Tax-mediated transcription of both constructs. We also found that treatment with sodium orthovanadate, a tyrosine phosphatase inhibitor, reduced Tax-mediated activation of both plasmids. These findings indicated that specific serine/threonine phosphorylation events are required for Tax-mediated HTLV-I LTR activation and also suggested that phosphorylation at tyrosine residues is involved in this process.
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PMID:Tax-induced HTLV-I LTR transcriptional activation is modulated by phosphorylation. 799 95

The periplasmic Escherichia coli cyclophilin is distantly related to human cyclophilin (34% sequence identity). Peptidyl-prolyl isomerase activity, cyclosporin A binding, and inhibition of the calcium-dependent phosphatase calcineurin are compared for human and E. coli wild-type and mutant proteins. Like human cyclophilin, the E. coli protein is a cis-trans peptidyl-prolyl isomerase. However, while the human protein binds cyclosporin A tightly (Kd = 17 nM), the E. coli protein does not (Kd = 3.4 microM). The mutant F112W E. coli cyclophilin has enhanced cyclosporin binding (Kd = 170 nM). As for the human protein, the complex of the E. coli mutant with cyclosporin A inhibits calcineurin. Here we describe the structure at pH 6.2 of cyclosporin A bound to the mutant E. coli cyclophilin as solved with solution NMR methods. Despite the low overall sequence identity, the structure of the bound cyclosporin A is virtually identical in both proteins. To assess differences of the cyclosporin binding site, the solution structure of wild-type E. coli cyclophilin was compared with structures of uncomplexed human cyclophilin A and with cyclosporin bound. Despite the structural similarity of bound cyclosporin A, the architecture of the binding site in the E. coli protein is substantially different at the site most distant to tryptophan 121 (human sequence). This site is constructed by a five-residue insertion in a loop of the E. coli protein, replacing another loop in the human protein.
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PMID:The mutant Escherichia coli F112W cyclophilin binds cyclosporin A in nearly identical conformation as human cyclophilin. 818 Jan 97

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 immunosuppressive cyclic nonapeptide cyclolinopeptide A inhibits calcium-dependent, but not calcium-independent, activation of T lymphocytes comparably to the actions of cyclosporin A and FK506. The concentration required for complete inhibition, however, is 10 times higher than that of cyclosporin A. In addition, we demonstrate that calcineurin, a phosphatase which plays an important role in T lymphocyte signalling, is inhibited in vitro by cyclolinopeptide A by a mechanism dependent on the peptidyl-prolyl cis-trans isomerase (PPIase) cyclophilin A but not FKBP12. Direct binding of cyclolinopeptide A to cyclophilin A was confirmed using tryptophan fluorescence studies and PPIase assays. These results represent a third example of the production of a natural product that neutralises calcineurin by a mechanism dependent on the primary binding to a PPIase.
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PMID:Cyclolinopeptide A (CLA) mediates its immunosuppressive activity through cyclophilin-dependent calcineurin inactivation. 941 31


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