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 major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.
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PMID:Human prostatic acid phosphatase has phosphotyrosyl protein phosphatase activity. 301 2

When the immunosuppressants cyclosporin A (CsA) and FK506 bind to their intracellular receptors (immunophillins), they form complexes that bind to calcineurin and block calcineurin-dependent signaling pathways in immune cells. Previously, we reported that higher plants also express immunophilins and have a Ca(2+)-dependent signaling pathway sensitive to immunophilin-ligand complexes. Based on an N-terminal peptide sequence of a chloroplast-localized cyclophilin (pCyP B), we isolated a cDNA clone encoding the preprotein of the cyclophilin. The deduced amino acid sequence of this cDNA starts with a putative transit sequence for chloroplast targeting. The mature pCyP B protein has rotamase activity with low-substrate specificity. Enzyme activity was inhibited by CsA with an inhibition constant of 3.9 nM. Similar to other CyPs from mammalian cells, pCyP B, when complexed with CsA, inhibited the phosphatase activity of bovine calcineurin. The mRNA level of pCyP B was high in leaf tissue but was not detectable in roots. Expression of the transcript in the leaf tissues was regulated by light and induced by heat shock. These findings illustrate the conserved nature of cyclophilin proteins among all of the eukaryotes and suggest that cyclophilins have a unique mode of regulation in higher plants.
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PMID:pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean. 806 22

The cDNA encoding human brain protein phosphatase inhibitor-1 (I-1) was expressed in Escherichia coli. Following PKA phosphorylation at a threonine, recombinant human I-1 was indistinguishable from rabbit skeletal muscle I-1 as a potent and specific inhibitor of the type-1 protein serine/threonine phosphatase (PP1). N-Terminal phosphopeptides of I-1 that retained the selectivity of intact human I-1 highlighted a functional domain that mediates PP1 inhibition. Substituting alanine in place of threonine-36 eliminated I-1 phosphorylation by PKA and its phosphatase inhibitor activity. An acidic residue was substituted in place of the phosphoacceptor to produce I-1(T35D), a constitutive phosphate inhibitor. I-1(T35D) was an equally effective inhibitor of PP1 and the type-2 phosphatase, PP2A. However, CNbr digestion of I-1(T35D) yielded an N-terminal peptide that showed 100-fold increased specificity as a PP1 inhibitor. This provided new insight into a unique conformation of the phosphorylated I-1 that accounts for selective inhibition of PP1 activity. Truncation of an active I-1 phosphopeptide identified an N-terminal sequence that was reduced in addition to threonine-35 phosphorylation to inhibit PP1 activity. Biosensor studies demonstrated that PP1 bound to both Phosphorylated and dephosphorylated I-1 and suggested that distinct elements of I-1 structure accounted for PP1 binding and inhibition. Our data point to multiple interactions between the I-1 functional domain. and the PP1 catalytic subunit that define this phosphoprotein as a physiological regulator of the type-1 protein phosphatase.
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PMID:Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase-1 inhibitor. 861 7

In response to stimulation of B-cells through cell surface IgM, the activity of the serine/threonine protein phosphatase PP1, but not PP2A, was transiently decreased and reached a minimum 10-20 min after the stimulation. The decrease was more profound in the immature B-cell line WEHI-231, than in the mature B-cell line BAL-17. Under these conditions, PP1alpha, an isoform of PP1, showed unique alterations in the patterns of several spots with distinct isoelectic points in the Western blot after two-dimensional electrophoresis, whereas another isoform, PP1delta, did not show any alteration. PP1gamma1 and PP1gamma2 were not detected in B-cells. Similar alterations in these spots were observed in B-cells stimulated by PMA. When partially purified PP1 consisting of PP1alpha and PP1delta was incubated with [gamma-32P]ATP and PKC, radioactive spots of PP1alpha could be detected, but no spot of PP1delta was detected. Because differences in sequence among PP1 isoforms are mostly restricted to their C-terminals, phosphorylation rates of the C-terminal peptides containing the PKC-phosphorylation motif were compared. The C-terminal peptide of PP1alpha is a better substrate for PKC than those of PP1gamma1 and PP1gamma2, and is phosphorylated at the serine residue corresponding to Ser-325 of PP1alpha. The corresponding C-terminal region of PP1delta does not contain the phosphorylation site. On the other hand, there was a large difference in subcellular distribution of PP1delta, but not PP1alpha, between immature and mature B-cells. From these results, it was strongly suggested that PP1alpha is involved, via phosphorylation by PKC, in the regulation of signal transduction in response to the stimulation of B-cells through cell surface IgM.
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PMID:Alterations in type-1 serine/threonine protein phosphatase PP1alpha in response to B-cell receptor stimulation. 939 75

Posttranslational modification of proteins by kinases and phosphatases plays an important role in the regulation of cellular signaling in general and neurochemistry in particular. This also applies to vertebrate photoreceptors where phosphorylation of rhodopsin causes uncoupling from the signal transduction cascade. Functional activity of rhodopsin is restored after substitution of the bleached photopigment 11-cisretinal and by dephosphorylation of the opsin moiety. Phosphatases type-1 and type-2A have been identified in vertebrate retinae. Recently, we have shown by molecular cloning that two isozymes of protein phosphatase type-2C (PP2C, PPM) do exist in retinal tissue. In this report, we have purified PP2Calpha and PP2Cbeta from bovine retinae. Thirty to 40% of PP2C was recovered in the cytosolic fraction. Biochemical properties of native and heterologously expressed recombinant enzymes were similar. Enzymatic activity is strictly dependent on the presence of Mg2+. Addition of Ca2+ ions inhibits Mg2+-sustained activity. Antiserum raised against a C-terminal peptide of PP2Cbeta specifically labeled the outer segments of rod photoreceptor cells. PP2C protein levels were significantly reduced in RCS rats, which develop age-dependent photoreceptor degeneration comparable to the hereditary disease retinitis pigmentosa. Although the retinal substrate(s) remain to be identified, the results suggest that PP2C modulates cellular components of the phototransduction machinery.
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PMID:Protein phosphatase type-2C isozymes present in vertebrate retinae: purification, characterization, and localization in photoreceptors. 948 68

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2.
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PMID:Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family. 969 11

In the investigation of the sequences of myosin phosphatase target subunit 1 (MYPT1) involved in binding the substrate and catalytic subunit of protein phosphatase type 1 (PP1c), fragments of MYPT1 were prepared and characterized. The shortest fragment capable of full activation of PP1c contained the sequence of residues 1-295. Within this fragment, the N-terminal sequence of residues 1-38 is involved in activation of PP1c (kcat) and the ankyrin repeats (residues 39-295) were involved in substrate binding (Km). The ankyrin repeats alone (residues 39-295) and the C-terminal fragment of residues 667-1004 did not activate PP1c. Using gel filtration, an interaction with PP1c was detected for the sequences of residues 1-295, 17-295, and 1-170. Affinity columns were prepared with various fragments to assess binding of PP1c. Binding to the column with residues 1-295 was strongest, followed by the binding to the column with residues 1-170. A weak interaction was observed with the column with residues 1-38. The column with residues 1-295 was used to isolate PP1c from gizzard. The purified PP1c was activated by MYPT1 and fragments to a greater extent than previous preparations. These results suggest that the N-terminal sequence (residues 1-38) and the ankyrin repeats are involved in binding PP1c. The C-terminal ankyrin repeats appear to be dominant, but there is an interaction of PP1c with the N-terminal ankyrin repeats. The N-terminal peptide has two apparent functions, the binding of PP1c via the consensus binding sequence and activation of PP1c by the sequence of residues 1-16.
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PMID:Interaction of myosin phosphatase target subunit 1 with the catalytic subunit of type 1 protein phosphatase. 984 38

Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated. 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase C at serine 652 and thereby inactivated. Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme. When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylated and inactivated. Under in vivo conditions, two phosphate residues were incorporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1. The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase. The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway. Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte.
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PMID:Phosphorylation and inactivation of yeast 6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypotonic stress. 1172 81

Secretory granules of chromaffin cells contain catecholamines and several antimicrobial peptides derived from chromogranins and proenkephalin-A. These peptides are secreted in the extracellular medium following exocytosis. Here, we show that ubiquitin is stored in secretory chromaffin granules and released into the circulation upon stimulation of chromaffin cells. We also show that the C-terminal fragment (residues 65-76) of ubiquitin displays, at the micromolar range, a lytic antifungal activity. Using confocal laser scan microscopy and rhodamine-labeled synthetic peptides, we could demonstrate that the C-terminal peptide (residues 65-76) is able to cross the cell wall and the plasma membrane of fungi and to accumulate in fungi, whereas the N-terminal peptide (residues 1-34) is stopped at the fungal wall level. Furthermore, these two peptides act synergistically to kill filamentous fungi. Because of the interaction of the C-terminal sequence of ubiquitin with calmodulin, the synthetic peptide (residues 65-76) was tested in vitro against calmodulin-dependent calcineurin, an enzyme crucial for fungal growth. This peptide was found to inhibit the phosphatase activity of calcineurin. Our data show a new property of ubiquitin C-terminal-derived peptide (65-76) that could be used with N-terminal peptide (1-34) as a new potent antifungal agent.
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PMID:The N- and C-terminal fragments of ubiquitin are important for the antimicrobial activities. 1259 74

The important role of the serine/threonine protein phosphatase 2A (PP2A) in various cellular processes requires a precise and dynamic regulation of PP2A activity, localization, and substrate specificity. The regulation of the function of PP2A involves the reversible methylation of the COOH group of the C-terminal leucine of the catalytic subunit, which, in turn, controls the enzyme's heteromultimeric composition and confers different protein recognition and substrate specificity. We have determined the structure of PPM1, the yeast methyltransferase responsible for methylation of PP2A. The structure of PPM1 reveals a common S-adenosyl-l-methionine-dependent methyltransferase fold, with several insertions conferring the specific function and substrate recognition. The complexes with the S-adenosyl-l-methionine methyl donor and the S-adenosyl-l-homocysteine product and inhibitor unambiguously revealed the co-substrate binding site and provided a convincing hypothesis for the PP2A C-terminal peptide binding site. The structure of PPM1 in a second crystal form provides clues to the dynamic nature of the PPM1/PP2A interaction.
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PMID:Structure of protein phosphatase methyltransferase 1 (PPM1), a leucine carboxyl methyltransferase involved in the regulation of protein phosphatase 2A activity. 1466 May 64


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