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)

Calmodulin (CaM) mediates the Ca(2+)-dependent activation of many enzyme systems in accordance with its cellular localization. We have described previously a muscarinic receptor-mediated translocation of CaM from membranes into the cytosol of SK-N-SH human neuroblastoma cells. To explore the potential targets (CaM-binding proteins, CaMBP) for CaM upon translocation, a photoreactive CaM derivative was introduced into living SK-N-SH cells using a scrape-loading technique. Scrape-loading incorporated rhodamine isothiocyanate-labeled CaM with an efficiency of 38%. CaM-diazopyruvamide (CaM-DAP), a Ca(2+)-dependent and CaM-specific probe, was also introduced into the cells. The muscarinic agonist carbachol stimulated a translocation of CaM from membranes into cytosol in CaM-DAP-loaded SK-N-SH cells. Upon photochemical cross-linking, cross-linked adducts of CaM-CaMBP were detected by immunoblotting with anti-CaM antibody. Carbachol stimulated increased photoaffinity labeling of three proteins with relative adduct molecular masses of 70, 120, and 180 kDa. The time course of labeling for the 70- and 120-kDa adducts showed maximal increased by 15-30 min. The 180-kDa adduct displayed a slower time course of maximal labeling, with increases maintained for 2-4 h. Subtracting the molecular mass of CaM, carbachol stimulated binding to CaMBPs of 55, 105, and 163 kDa. Predominant cellular CaMBP were identified using a biotinylated CaM overlay procedure. Western blot analysis indicated the expression of specific CaM-dependent enzymes such as calcineurin, phosphodiesterase, the beta-isoform (rat brain) of CaM kinase II, and Ca(2+)-ATPase. Numerous cytoskeletal CaMBP were expressed such as microtubule-associated protein-2, spectrin, tubulin, caldesmon, adducin, and neuromodulin. Of the CaMBP expressed, phosphodiesterase, calcineurin, caldesmon, and adducin cross-linked with CaM-DAP in the loaded SK-N-SH cells. Carbachol stimulated the time-dependent CaM-DAP labeling of calcineurin and adducin. This study demonstrates the novel incorporation of a photoreactive CaM derivative into living cells, as well as muscarinic receptor-activated CaM-DAP interaction with several cellular CaMBP. We postulate that carbachol-stimulated CaM translocation in SK-N-SH cells may affect the activity of CaM-dependent enzymes and may alter aspects of cytoskeletal function.
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PMID:Carbachol stimulates binding of a photoreactive calmodulin derivative to calmodulin-binding proteins in intact SK-N-SH human neuroblastoma cells. 155 1

Hippocampal slices were preincubated with 32P-orthophosphate and used to study the effect of glutamate analogs on protein phosphorylation. NMDA induced a rapid, 70% decrease in the phosphorylation of the microtubule-associated protein MAP2, with no change in the total amount of MAP2. Both competitive and noncompetitive NMDA antagonists blocked the effect of NMDA, but a glutamate antagonist acting at non-NMDA receptors did not. Kainate and quisqualate were less potent than NMDA in stimulating dephosphorylation of MAP2. Other forebrain regions (necortex, striatum, and olfactory bulb) also showed dephosphorylation of MAP2 in response to NMDA. These and other results suggest that NMDA receptor activation induces the dephosphorylation of MAP2 by stimulating a protein phosphatase, possibly the calcium/calmodulin-dependent protein phosphatase calcineurin. Moreover, they indicate that alteration in the properties of a microtubule-associated protein may account for some of the effects of glutamate on postsynaptic neurons.
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PMID:Activation of NMDA receptors induces rapid dephosphorylation of the cytoskeletal protein MAP2. 216 65

Concurrent exposures to organophosphorus insecticide leptophos and the industrial solvents n-hexane and toluene were implicated in causing an outbreak of neuropathy in workers. Although both leptophos and n-hexane produce central-peripheral distal axonopathy, the morphology and distribution of neuropathic lesions are distinct, reflecting different modes of action. The molecular mechanisms of organophosphorus compound-induced delayed neurotoxicity (OPIDN) and aliphatic hexacarbon-induced neurotoxicity have been investigated utilizing various biochemical techniques, (i.e. one- and two-dimensional gel electrophoresis, immunoblotting, peptide mapping). Oral administration of tri-o-cresyl phosphate (TOCP) produced delayed neurotoxicity and increased in vitro Ca2+ and calmodulin-dependent kinase protein phosphorylation of cytoskeletal proteins in brain, spinal cord, and sciatic nerve of chickens. This enhanced protein phosphorylation correlated well with the following characteristics of OPIDN: test chemical, whether an OPIDN-producing or not; dose-dependence and time course of the effect; and the animal sex sensitivity, age selectivity, and species susceptibility. The proteins that showed an increased phosphorylation were identified to be; alpha- and beta-tubulin, microtubule-associated protein-2 (MAP-2), and the 3 neurofilament proteins 70 kDa, 160 kDa, and 210 kDa. Further studies suggested that the increased protein phosphorylation is not related to an effect on protein phosphatase or ATPase activity, but rather to altered Ca2+-calmodulin kinase II activity. Aliphatic hexacarbon-induced neurotoxicity is characterized by an accumulation of 10 nm neurofilaments above the nodes of Ranvier in the spinal cord and peripheral nerve. Treatment of rats with 2,5-hexanedione, the active neurotoxic metabolite of n-hexane, produced protein crosslinking in a dose-dependent manner. This treatment also decreased protein phosphorylation of neurofilament proteins as well as MAP-2. These studies demonstrate the involvement of cytoskeletal proteins in the molecular pathogenesis of chemical-induced neurotoxicity.
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PMID:Cytoskeletal proteins as targets for organophosphorus compound and aliphatic hexacarbon-induced neurotoxicity. 283 76

Ribosomal protein S6 is a component of the eukaryotic 40S ribosomal subunit that becomes phosphorylated on multiple serine residues in response to a variety of mitogens, including insulin, growth factors, and transforming proteins of many oncogenic viruses. Recently, an activated S6 kinase (S6 K II) has been purified to homogeneity from Xenopus eggs, and characterized immunologically and at the molecular level. Purified S6 K II can be deactivated in vitro by incubation with either protein phosphatase 1 or protein phosphatase 2A. Reactivation and phosphorylation of S6 K II occurs in vitro with an insulin-stimulated microtubule-associated protein-2 (MAP-2) protein kinase which is itself a phosphoprotein that can be deactivated by protein phosphatase 2A. These studies suggest that a step in insulin signalling involves sequential activation by phosphorylation of at least two serine/threonine protein kinases.
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PMID:Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. 284 85

A protein phosphatase has been isolated from brain using, as assay substrate throughout the purification, microtubule-associated protein-2 which had been phospho-labeled by its associated kinase. In contrast to other protein phosphatases, this phosphatase can effectively release phosphate from both the microtubule-binding and projection domains of microtubule-associated-protein-2. This enzyme appears to be a distinct, specific phosphatase that does not readily fit into previous classification schemes and is possibly the enzyme responsible for dephosphorylating microtubule-associated protein-2 in vivo.
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PMID:A brain phosphatase with specificity for microtubule-associated protein-2. 301 98

The most conspicuous brain microtubule-associated protein, MAP-2, has been shown to contain 8-10 mol of covalently bound phosphate/mol, as isolated. The MAP-2-associated cAMP-dependent protein kinase can add 10-12 more phosphates, using cycled microtubule preparations, but it does not catalyze exchange between ATP and the pre-existing protein phosphate. We now show that the phosphates that turn over in vivo, after intracerebral injection of 32Pi, are primarily in the projection domain of MAP-2, whereas the sites phosphorylated in vitro are more concentrated in the binding domain. Phosphoserine and phosphothreonine were recovered in a 6:1 ratio from partial acid hydrolysates of MAP-2 phosphorylated either in vivo or in vitro. A protein phosphatase, purified from brain, released residues from in vitro sites in both domains. The enzyme did not release appreciable phosphate that had turned over in vivo, and similar specificity was shown by three other purified protein phosphatases: calcineurin (also from brain) and smooth muscle phosphatases I and II. Thus, even in the projection domain, different sites may be involved.
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PMID:The sites at which brain microtubule-associated protein 2 is phosphorylated in vivo differ from those accessible to cAMP-dependent kinase in vitro. 398 Apr 81

A microtubule-associated protein (the 270-kDa MAP-2) was prepared in two defined states of phosphorylation by (a) phosphorylation by associated kinase to the extent of 11-14 mol/mol, and (b) removal of 70-80% of this phosphate with a protein phosphatase purified from brain. The newly introduced phosphate was in addition to about 10 mol/mol already present in MAP-2 as isolated; these phosphates were not appreciably released by the phosphatase and did not exchange with ATP. In microtubules assembled with phosphorylated (24 mol/mol) MAP-2 the assembly rate was decreased, microtubule length and critical concentration for assembly were unaffected, and rates of loss of subunits were increased from both microtubule ends. Phosphorylation also reduced the binding of MAP-2 to taxol-stabilized microtubules. These changes were unequivocally due to phosphorylation, since phosphatase treatment reversed all of them. The brain phosphatase used in these experiments was purified 3000-fold towards histone, but only 100-fold towards MAP-2, suggesting brain may contain another enzyme more specific for MAP-2. Calcineurin, however, had only a low activity for MAP-2.
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PMID:Microtubule assembly using the microtubule-associated protein MAP-2 prepared in defined states of phosphorylation with protein kinase and phosphatase. 614 Jan 63

Tau is a neuron-specific, microtubule-associated protein that forms paired helical filaments (PHFs) of Alzheimer's disease when aberrantly phosphorylated. We have attempted to elucidate the protein kinases and phosphatases that regulate tau phosphorylation. Incubation of rat, human, and rhesus monkey temporal neocortex slices with the phosphatase inhibitor okadaic acid induced epitopes of tau similar to those found in PHFs. Okadaic acid (1-20 microM) induced variant forms of tau at 60-68 kDa, which were recognized by the monoclonal antibodies Alz-50 (in humans only) and 5E2 and two polyclonal antipeptide antisera, OK-1 and OK-2. The phosphorylation-sensitive monoclonal antibody Tau-1 failed to recognize the slowest mobility forms of tau after okadaic acid treatment. FK-520 (1-10 microM), a potent inhibitor of calcineurin activity, was tested in brain slices and found not to alter tau mobility. However, combinations of FK-520 (5 microM) and okadaic acid (100 nM) caused tau mobility shifts similar to those seen after 10 microM okadaic acid treatment; similar results were seen using the calcineurin-selective inhibitor cypermethrin. Treatment of human slices with 10 microM okadaic acid decreased both protein phosphatase 2A and calcineurin activity; FK-520 inhibited only protein phosphatase 2B activity. A proposed tau-directed kinase, 42-kDa mitogen-activated protein kinase (p42mapk), was activated by okadaic acid (> 100 nM) but not FK-520 (5 microM). Nerve growth factor (100 ng/ml) activated p42mapk, particularly when used in combination with 100 nM okadaic acid; changes in tau mobility were seen when this kinase was activated. Forskolin (2 microM) antagonized the effects of nerve growth factor on both p42mapk activity and tau phosphorylation; forskolin alone had little effect on PHF-like tau formation induced by phosphatase inhibitors. These results outline complex interactions between tau-directed protein kinases and protein phosphatases and suggest potential sites for therapeutic intervention.
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PMID:Tau phosphorylation in brain slices: pharmacological evidence for convergent effects of protein phosphatases on tau and mitogen-activated protein kinase. 772 35

Mitochondria play an important role in modulating intracellular levels of calcium, and therefore compromised mitochondrial function often leads to disruptions in calcium homeostasis. In this study, the effects of two uncouplers of oxidative phosphorylation, carbonyl cyanide-3-chlorophenylhydrazone (CCCP) and p-trifluoromethoxyphenylhydrazone (FCCP), on calcium-mediated modifications of the microtubule-associated protein, tau, in rat brain slices were examined. Incubation of slices with CCCP or FCCP resulted in an increase in electrophoretic mobility of several of the tau isoforms, with no apparent loss of intact tau or the appearance of degradation products. These data indicated that disrupting mitochondrial function by dissipating the transmembrane potential resulted in the dephosphorylation of tau. This finding was confirmed by using a front phosphorylation assay to demonstrate a CCCP-induced decrease in the phosphorylation state of tau. The dephosphorylation of tau induced by the proton-ionophores appeared to be calcium-dependent since the effect was blocked by EGTA. In addition, the CCCP-induced dephosphorylation of tau was blocked by cyclosporin A, a selective inhibitor of the calcium-dependent phosphatase, calcineurin. These data strongly indicate that tau is a substrate for calcineurin in vivo. Finally, the levels of ATP were depleted to a similar extent in brain slices incubated in the presence of CCCP or CCCP and EGTA. These results demonstrated depletion of ATP alone was not sufficient to stimulate the dephosphorylation of tau in this experimental paradigm.
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PMID:Compromised mitochondrial function results in dephosphorylation of tau through a calcium-dependent process in rat brain cerebral cortical slices. 782 68

The ATP.Mg-dependent type-1 protein phosphatase activating factor (factor FA) was identified as a brain protein kinase that could phosphorylate microtubule-associated protein-2 (MAP-2) and thereby inhibit cross-linking interactions of MAP-2 with actin filaments and microtubules isolated from porcine brain. The phosphorylation sites were found to be equally located on both projection and microtubule-binding domains of MAP-2. Phosphoamino acid analysis revealed that the phosphorylation sites were on both serine and threonine residues, indicating that factor FA is a serine/threonine-specific MAP-2 kinase. Conversely, factor FA was further identified as a MAP-2 phosphatase activator that could promote the dephosphorylation of 32P-MAP-2 phosphorylated by factor FA itself and thereby potentiate cross-linking interactions of MAP-2 with actin and microtubules. Furthermore, the two opposing functions of factor FA can be selectively modulated in a reciprocal manner by pH change. For instance, alkaline pH could stimulate factor FA to work as a MAP-2 kinase but simultaneously block it to work as a MAP-2 phosphatase activator to potentiate the inhibition on the cross-linking interactions of MAP-2 with actin and microtubules. Taken together, the results provide initial evidence that a cyclic modulation of cross-linking interactions of MAP-2 with actin filaments and microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for rapid structural and functional regulation of neuronal cytoskeletal system.
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PMID:Cyclic modulation of cross-linking interactions of microtubule-associated protein-2 with actin and microtubules by protein kinase FA. 825 Oct 59


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