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 insulin receptor, purified from the hepatopancreas of the shrimp Penaeus monodon, is a hydrophobic heterodimer of subunits with relative masses (Mr) of 70,000 and 58,000, as estimated by FPLC on Superose 12 and SDS-PAGE. Only the subunit of Mr 70,000 was autophosphorylated after the addition of insulin. The autophosphorylation occurred specifically at Tyr residues, as demonstrated by the specific subsequent dephosphorylation by the phosphotyrosyl protein phosphatase from the hepatopancreas of the shrimp Penaeus monodon. Proteins of Mr 44,000 and Mr 32,000 on the plasma membrane from the hepatopancreas of the shrimp Panaeus monodon were phosphorylated by the autophosphorylated insulin receptor from the shrimp hepatopancreas, but not by that from the human placenta. The detergent, Triton X-100, caused noticeable enhancement of the autophosphorylation of both shrimp and human insulin receptors.
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PMID:Specific phosphorylation of membrane proteins of Mr 44,000 and Mr 32,000 by the autophosphorylated insulin receptor from the hepatopancreas of the shrimp Penaeus monodon (Crustacea: Decapoda). 840 97

Nucleoside diphosphate kinase (EC 2.7.4.6) (Ndk) is a ubiquitous enzyme functioning in the intracellular distribution of terminal phosphate bond energy among the various nucleotides used in synthetic and regulatory functions in cells. We have previously reported that in Pseudomonas aeruginosa, this important enzyme is transcriptionally regulated by the gene algR2 and posttranslationally regulated by a phosphoprotein phosphatase for the phosphorylated form of Ndk. We report here that an intracellular protease cleaves the 16-kDa form of Ndk to a 12-kDa form that undergoes autophosphorylation with an efficiency almost identical to that of the 16-kDa form. The 12-kDa form was found to be predominantly associated with the P. aeruginosa cell membrane fraction, whereas the 16-kDa form was predominantly cytoplasmic. In the membrane-associated state, the 12-kDa form of Ndk was found to synthesize GTP in preference to other nucleoside triphosphates. The specificity toward GTP synthesis could be abolished by the addition of Tween 20 or Triton X-100. The activity itself could be abolished by the addition of anti-Ndk antibody to the assay mixture. The formation of the 12-kDa form of Ndk and its association with the cell membrane were found to be related to the growth stage of P. aeruginosa, with less than 1% of the 12-kDa Ndk detectable in the membrane fraction at early log phase in comparison with the levels present at late stationary phase.
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PMID:Two forms of the nucleoside diphosphate kinase of Pseudomonas aeruginosa 8830: altered specificity of nucleoside triphosphate synthesis by the cell membrane-associated form of the truncated enzyme. 860 47

The mechanism of dephosphorylation of multiphosphorylated proteins in the brain is not well understood. We have used the multiphosphorylated protein, phosvitin as a model substrate and undertaken the purification and characterization of brain phosphatases that preferentially dephosphorylate multiphosphorylated proteins. Two phosvitin phosphatase activities, termed Phosvitin Phosphatase 1 and 2 (PvP1, PvP2), which show acidic pH optima were resolved from the 33,000g supernatant fraction from rat brain by a procedure employing successive DEAE-cellulose, Sepharose 6B, second DEAE-cellulose and FPLC/Superose 6 chromatography steps. Following FPLC/Superose 6 size exclusion chromatography of PvP1 and PvP2, single peaks of phosvitin phosphatase activities were eluted in the range of 160-220 kDa with acidic pH optima. When FPLC/Sepharose 6 chromatography was performed in the presence of 0.5 M NaCl and 0.1% Triton X-100, low molecular mass protein phosphatase forms were produced in addition to the high-M, activity peak, ranging from 25 to 35 kDa (PvP1) and from 15 to 25 kDa (PvP2). Under these conditions, both high- and low-M, forms of PvP1 and PvP2 exhibited neutral pH optima. Both phosphatases dephosphorylate also (i) phosphorylase a, (ii) the alpha and beta subunits of phosphorylase kinase, and (iii) the microtubule-associated protein tau, phosphorylated by cAMP-dependent protein kinase. The present results suggest that two forms of protein phosphatases, displayed molecular and biochemical characteristics both similar and distinct from type 1 and type 2A protein phosphatases, are present in rat brain.
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PMID:Partial purification and characterization of two phosvitin phosphatases from rat brain. 862 49

Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+ or in a medium containing the Ca2+ ionophore A23187. This Ca2+-induced dephosphorylation was calmodulin-dependent, suggesting that calcineurin was involved. In this regard, the calcineurin inhibitor deltamethrin inhibited the Ca2+ ionophore-induced dephosphorylation. A limited number of Mr 80,000-105,000 polypeptides were the most prominent phosphotyrosine-containing proteins present in human sperm. Unlike mouse sperm, which contains a tyrosine-phosphorylated isoform of hexokinase, a phosphotyrosine-containing hexokinase in human sperm was not detected. Most of the tyrosine-phosphorylated proteins were Triton X-100-insoluble and were localized to the principal piece of the flagellum, the region where the cytoskeletal fibrous sheath is found. Prominent phosphotyrosine-containing proteins of Mr 82,000 and 97,000 were identified as the human homologues of mouse sperm AKAP82, the major fibrous sheath protein, and pro-AKAP82, its precursor polypeptide, respectively. These proteins are A Kinase Anchor Proteins, polypeptides that sequester protein kinase A to subcellular locations. Taken together, these results suggest that protein tyrosine phosphorylation may be part of a signal transduction cascade(s) regulating events pertaining to capacitation and/or motility in mammalian sperm and that an interrelationship between tyrosine kinase and cAMP signaling pathways exists in these cells.
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PMID:Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. 894 91

Neuronal nuclear fraction N1 was isolated from cerebral cortices of 15-day-old rabbits, and nuclear subfractions prepared, in order to study the location of nuclear lyso platelet-activating factor (lyso-PAF) acetyltransferase and alkylglycerophosphate (AGP) acetyltransferase, and factors that affect the loss of these two nuclear activities. Subfractionation of prelabelled N1 indicated that the nuclear envelope had the highest percentage of the radioactive acetylated products alkylacetylglycerophosphate (AAGP) and PAF, and the distribution of these phospholipids reflected phospholipid distributions in the nuclear subfractions. The majority (95%) of radioactive AAGP and PAF was also recovered in Triton X-100 extracts of prelabelled nuclei, suggesting that these acetylated lipids are located in nuclear membranes rather than in the nuclear matrix/chromatin. Of the nuclear subfractions, the envelope had the highest AGP and lyso-PAF acetyltransferase specific activities which were close to corresponding values seen in the parent N1 fraction. Thus the nuclear AGP and lyso-PAF acetyltransferases were principally localized to the nuclear membranes. Differentials in activity loss were seen for the two acetyltransferase activities. In the nuclear envelope fractions, the lyso-PAF acetyltransferase was the more susceptible to oxidation reactions which could be reversed or blocked by the use of reducing agents. In preincubations, N1 showed greater losses in lyso-PAF acetyltransferase activity than in AGP acetyltransferase activity, losses which were not attributable to oxidation. Addition of cytosolic fraction S3 to preincubations promoted losses for each acetyltransferase in N1, and gave evidence for cytosolic and endogenous nuclear contributions to the activity loss. Addition of okadaic acid to the preincubations did not prevent the decline of either acetyltransferase in intact nuclei, but did diminish the loss of nuclear lyso-PAF acetyltransferase activity promoted by S3 addition, and also blocked the loss of this acetyltransferase seen in preincubations of isolated nuclear envelopes. This suggests that nuclear lyso-PAF acetyltransferase is susceptible to okadaic acid-sensitive nuclear and cytosolic protein phosphatase activities, while AGP acetyltransferase may lose activity by the action of other phosphatases or by other mechanisms within the nucleus.
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PMID:Neuronal nuclear acetyltransferases involved in the synthesis of platelet-activating factor are located in the nuclear envelope and show differential losses in activity. 910 99

We previously reported that activation of protein kinase A in cultured rat dorsal root ganglion neurons, treated concomitantly with low concentrations of okadaic acid that selectively inhibit protein phosphatase-2A, enhanced the Triton X-100 solubility of neurofilament triplet proteins. We now show that peripherin and alpha-internexin follow the same fragmentation profile as the neurofilament subunits, consistent with the notion that all five cytoplasmic intermediate filament proteins in these neurons form an integrated filamentous network whose assembly can be modulated by protein kinase A. Similar to the situation previously observed for the light neurofilament subunit, there was a strong correlation between phosphorylation of the amino-terminal head domain of peripherin and filament fragmentation. In contrast, insignificant levels of 32P were incorporated into alpha-internexin under conditions promoting disassembly, indicating that phosphorylation of this protein is not involved directly in filament fragmentation. The situation for the mid-sized neurofilament subunit (NFM) was not as clear-cut. Phosphopeptide mapping of NFM revealed many head and tail domain phosphorylation sites. However, changes in NFM head domain phosphorylation under conditions promoting filament disassembly were not as pronounced as for peripherin.
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PMID:Intermediate filament disassembly in cultured dorsal root ganglion neurons is associated with amino-terminal head domain phosphorylation of specific subunits. 957 70

Low Mr phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet-derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X-100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low Mr PTP overexpression.
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PMID:Low molecular weight phosphotyrosine protein phosphatase translocation during cell stimulation with platelet-derived growth factor. 972 Sep 13

The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.
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PMID:Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells. 977 56

A rapid and gentle procedure for preparing demembranated, cytosol-free sperm models was applied to fowl spermatozoa. Intact spermatozoa were introduced to a Triton X-100-containing extraction medium layered on top of a discontinuous Percoll gradient in a 1.5 ml microfuge tube. After brief exposure to the extraction medium, spermatozoa were separated from the plasma membrane and detergent-soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they were recovered. Optimum conditions consisted of a Triton X-100 concentration in the extraction medium of 0.15%, duration of demembranation time of 1.5 min and ATP concentration in the reactivation medium of 0.5 mM. Demembranated sperm models obtained by this procedure could be reactivated, and the motility at 30 degrees C was more than 60%, but negligible at 40 degrees C. These values were similar to those obtained from the conventional method, in which centrifugation is not carried out, and which results in some of the cytosolic components being transferred to the reactivation medium along with the spermatozoa. Inhibition of motility was observed following the addition of EGTA or myosin light chain kinase (MLCK) substrate peptide at 30 degrees C, whilst the presence of protein phosphatase inhibitors, such as calyculin A or okadaic acid, permitted the restoration of motility at 40 degrees C. These results demonstrate that the axoneme and/or accessory cytoskeletal components are directly involved in the temperature-dependent regulatory system of fowl sperm motility in the absence of plasma membrane and/or soluble components of cytoplasm.
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PMID:Temperature-dependent flagellar motility of demembranated, cytosol-free fowl spermatozoa. 988 71

Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.
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PMID:Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors. 1079 19


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