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)

Abnormally hyperphosphorylated tau is the major protein subunit of paired helical filaments in Alzheimer brains. We have examined its site-specific dephosphorylation by different protein phosphatases. Dephosphorylation of tau was monitored by its interaction with several phosphorylation-dependent antibodies. Alzheimer tau was dephosphorylated by brain protein phosphatase-2B at the abnormally phosphorylated sites Ser46, Ser199, Ser202, Ser235, Ser396, and Ser404, and its relative mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis shifted to that of normal tau. Protein phosphatases-1 and -2A could dephosphorylate only some of the above six phosphorylation sites. These results indicate that protein phosphatase-2B might be involved in hyperphosphorylation of tau in Alzheimer's disease.
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PMID:Alzheimer's disease abnormally phosphorylated tau is dephosphorylated by protein phosphatase-2B (calcineurin). 829 42

Cyclosporin A, a calcineurin inhibitor, was administered into the rat hippocampus. Seven days after drug administration the effects of phosphatase activity suppression in the brain to changes in neuronal cytoskeletal proteins were studied. Rat brain homogenates injected with cyclosporin A showed 16-38% suppression of Ca/calmodulin-dependent phosphatase activity measured by 32P released from 32P-labeled histone, indicating that cyclosporin A acts as an inhibitor of calcineurin after binding with cyclophilin in the brain. Hematoxylin-eosin staining revealed many basophilic neurons in the pyramidal layer of hippocampus, cerebellum, thalamus and cerebral cortex. Immunohistochemical study with anti-phosphorylated neurofilament 200kDa antibody showed positive staining of neuronal perikarya of these basophilic neurons. The number of immunopositive neurons with anti-phosphorylated neurofilament 200KDa increased with the concentration of cyclosporin A injected into the brain, indicating a dose-dependent effect of the compound. Staining with anti-dephosphorylated neurofilament 200KDa (SMI-32) was decreased in neuronal perikarya of cyclosporin A injected brains compared with that of control brains injected with dimethyl sulfoxide alone, suggesting an increased phosphorylation of neurofilament 200KDa subunit protein. The perikarya of these basophilic neurons were not stained with anti-PHF (paired helical filaments) or anti-tau antibodies. Immunostaining with anti-ubiquitin was not increased in these cells. Immunostaining with anti-calcineurin A and anti-calcineurin B showed positive staining of neurons in hippocampus, cerebellum and caudate nucleus both in experimental and control brains. Calcineurin B immunoreactivity was more intense in pyramidal cells in hippocampus injected with cyclosporin A than in controls. Western blot study with antibody against calcineurin A revealed significantly more degradation products of calcineurin A in cyclosporin A injected brains. The present data suggest that cyclosporin A inhibits calcineurin activity in the brain, which results in increased phosphorylation of perikaryal neurofilaments. Since abnormal phosphorylation is speculated in the pathological process of Alzheimer's disease, the results will help elucidate the participation of phosphatase in the pathology of cytoskeletal proteins in Alzheimer's disease.
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PMID:Phosphorylated neurofilament accumulation in neuronal perikarya by cyclosporin A injection in rat brain. 838 21

Microtubule-associated protein tau is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of tau to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated tau could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of tau might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used 32P-labeled phosphorylase kinase and poly(Glu, Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/phosphothreonyl-protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl-protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age-matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of tau in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of beta-amyloid by augmenting the amyloidogenic pathway processing of beta-amyloid precursor protein.
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PMID:Phosphoprotein phosphatase activities in Alzheimer disease brain. 839 66

Hyperphosphorylated forms of the microtubule-associated protein tau are components of the paired helical filaments (PHFs) seen in patients with Alzheimer's disease. Slices of human lateral temporal cortex were obtained from tissues removed incidental to resections for intractable hippocampal epilepsy. Tau phosphorylation in temporal lobe slices was determined using mobility shifts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection with the monoclonal antibodies Alz-50, 5E2, and Tau-1. The results indicate that tau phosphorylation was altered in a dose-dependent manner by the phosphatase inhibitor okadaic acid, but not by N-methyl-D-aspartate, quisqualate, or kainate. The slowest mobility forms of tau, termed "PHF-like tau," produced by okadaic acid treatment were dephosphorylated by purified protein phosphatase 2B (calcineurin). Formation of PHF-like tau peptides was blocked by KN-62, 1[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, an inhibitor of Ca2+/calmodulin-dependent protein kinase II. The protein kinase inhibitor staurosporine also prevented formation of PHF-like tau. These data suggest that phosphorylation of tau is regulated by Ca(2+)-dependent protein kinases and okadaic acid-sensitive protein phosphatases, alterations of which may be implicated in the pathogenesis of Alzheimer's disease.
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PMID:Okadaic acid induces hyperphosphorylated forms of tau protein in human brain slices. 849 35

We have reported that many sites of tau in fetal brain (fetal-tau) as well as in paired helical filaments (PHF-tau) are phosphorylated. In the present study, we used site-specific antibodies and peptide mapping to examine protein phosphatases involved in dephosphorylation of fetal-tau and PHF-tau. Immunoblot analysis and electrophoretic mobility showed that protein phosphatases 1 and 2A and calcineurin could dephosphorylate fetal-tau and PHF-tau. Phosphoserines 199, 202, 396, and 413 and phosphothreonine 231, numbered according to the longest human tau isoform, were dephosphorylated, as shown by the immunoblot analysis. Phosphoserine 422 was dephosphorylated by protein phosphatase 2A and calcineurin, but not by protein phosphatase 1. Peptide mapping with Achromobacter lyticus protease 1 showed that phosphoserines 199, 202, 235, and 396 and phosphothreonine 231 were dephosphorylated by protein phosphatases. Fetal-tau was more rapidly dephosphorylated by protein phosphatase 2A and calcineurin than PHF-tau. Interestingly, PHF-tau which had not been solubilized with guanidine HCl was little dephosphorylated by protein phosphatases. Thus, PHF-tau in neurofibrillary tangles of Alzheimer's disease brain is likely to be resistant to dephosphorylation by protein phosphatases.
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PMID:Dephosphorylation of fetal-tau and paired helical filaments-tau by protein phosphatases 1 and 2A and calcineurin. 872 Jan 39

The microtubule-associated protein tau is more highly phosphorylated at certain residues in developing brain and in Alzheimer's disease paired helical filaments than in adult brain. We examined the regulation of tau phosphorylation at some of these sites in rat brain using the phosphorylation state-dependent anti-tau antibodies AT8, Tau1, and PHF1. The AT8 and PHF1 antibodies bind to phosphorylated tau, while Tau1 binds to unphosphorylated tau. Levels of tau reactive for AT8 were high only during the first postnatal week, with levels in adult declining to approximately 5% of the levels in neonates. In neonatal forebrain slices, tau became rapidly dephosphorylated at the AT8 and Tau1 sites during incubation at 34 degrees C, but was incompletely dephosphorylated at the PHF1 site. Dephosphorylation at AT8 sites, but not at Tau1 or PHF1 sites, was completely inhibited by 1 microM okadaic acid. Hence the regulation of tau phosphorylation by okadaic acid-sensitive phosphatase(s) was site-specific. Addition of 1 microM okadaic acid after dephosphorylation at the AT8 locus yielded a partial recovery of AT8 immunoreactivity, and incubation with basic fibroblast growth factor increased phosphorylation at the AT8 site in a dose-dependent manner. These results indicate that endogenously active and basic fibroblast growth factor stimulated tau kinases directed toward an Alzheimer's disease-related site were present in the slices. In adult brain slices, the small pool of AT8-reactive tau was remarkably insensitive to dephosphorylation during incubation, and okadaic acid treatment induced only small increases in AT8 immunoreactivity. These results suggest that tau phosphorylation in adult brain is less dynamic than in neonatal brain. These findings indicate that neonatal tau is not only phosphorylated more highly than adult tau, but also more dynamically regulated by protein phosphatases and protein kinases than adult tau. The inability of okadaic acid to induce large increases in tau phosphorylation in adult rat brain slices suggests that a loss of protein phosphatase activity alone would not be sufficient to produce the hyperphosphorylation observed in Alzheimer's disease paired helical filaments. Stimulation of kinase activity by basic fibroblast growth factor is likely to modulate tau function during development, and may contribute to the genesis of hyperphosphorylated tau in Alzheimer's disease.
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PMID:Site-specific regulation of Alzheimer-like tau phosphorylation in living neurons. 873 Jul 15

Neurofibrillary tangles (NFT), neuritic plaques, and dystrophic neurites are the classic neuropathologic hallmarks of Alzheimer's disease (AD), all of which contain to varying degrees abnormally and/or hyperphosphorylated forms of the microtubule-associated protein tau. Protein phosphatase 2B (calcineurin) dephosphorylates tau isolated from AD brains to control levels in vitro as well as regulates tau phosphorylation and function in vivo. It has been hypothesized that the changes in tau phosphorylation observed in AD may be due to increases in kinase activity and/or decreases in phosphatase activity. In order to investigate the latter possibility, we examined calcineurin enzyme activity using the substrate para-nitrophenyl-phosphate (pNPP) in postmortem brain samples from individuals with moderate to severe AD (n = 8) and age-matched controls (n = 7). The stimulation of calcineurin activity by manganese chloride (1 mM) was reduced by 60% (p < 0.01) in whole-cell homogenates prepared from AD temporal cortex (Brodmann area 38). On the other hand, in P2 membrane fractions, the stimulation of calcineurin activity by manganese chloride as well as nickel chloride (1 mM) was reduced by 37% (p < 0.05) and 79% (p < 0.01), respectively. The manganese-stimulated calcineurin activity in the temporal cortex inversely correlated with both the number of NFT (r = -0.60, p < 0.02) and neuritic/core plaques (r = -0.63, p < 0.02) in whole-cell homogenates, but only with NFT (r = -0.61, p < 0.02) in P2 membrane fractions. The nickel-stimulated calcineurin activity did not correlate with neuropathology measures in either whole-cell or P2 membrane fractions. In striate visual cortex (Brodmann area 17), an area relatively unaffected in AD, neither whole-cell nor P2 membrane calcineurin activity were significantly altered. To our knowledge, this is the first report of a reduction in calcineurin phosphatase activity in AD which correlates with the neuropathological features in a region-, subcellular fraction-, and divalent cation-specific manner.
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PMID:Reduction of calcineurin enzymatic activity in Alzheimer's disease: correlation with neuropathologic changes. 875 82

Microtubule associated protein tau promotes the assembly of microtubules by binding to microtubules and stabilizing their structure. In Alzheimer disease brain, tau is abnormally hyperphosphorylated and the altered tau is unable to promote the in vitro assembly of microtubules. In the present study, we found that dephosphorylation of abnormally phosphorylated tau by protein phosphatase-2A1, -2B or -1 restored its biological activity both in the nucleation and in the assembly of microtubules. Both the amount of phosphate released and the rate of restoration of microtubule assembly promoting activity of the abnormal tau were greater on dephosphorylation by protein phosphatase-2A1 than -2B or -1. During 90 min incubation at 37 degrees C protein phosphatase-2A1, -2B and -1 released respectively approximately 57%, approximately 36% and approximately 30% of tau phosphate. Association of the restoration of the biological activity of the abnormal tau dephosphorylated by different phosphatases and the immunochemical identification of the dephosphorylated sites revealed that Ser-235 is not critical in tau function, and that the Thr-231 is probably involved in the regulation of the nucleation and not the assembly of microtubules. These studies indicate that the phosphorylation of tau in situ might be regulated by protein phosphatase-2A, -2B and -1 and activation of these enzyme activities might arrest the Alzheimer neurofibrillary degeneration.
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PMID:Restoration of biological activity of Alzheimer abnormally phosphorylated tau by dephosphorylation with protein phosphatase-2A, -2B and -1. 879 8

Amyloid beta peptide (Abeta) is a short peptide that is the major constituent of the amyloid plaques and cerebrovascular amyloid deposits found in Alzheimer's disease. The lack of availability of a cell-free system in which to study Abeta formation has limited our understanding of the molecular mechanisms involved in its production. We report here the reconstitution of such a cell-free system. The reconstituted Abeta formation was temperature-dependent and required ATP. Preincubation with purified protein kinase C (PKC) induced a pronounced inhibition of Abeta formation, similar to that observed in intact cells upon stimulation of PKC. The calmodulin antagonists W-7 and trifluoperazine inhibited Abeta formation and enhanced the action of PKC in both the cell-free system and intact cells. A role for the calcium/calmodulin-activated protein phosphatase calcineurin in the regulation of Abeta formation was demonstrated using a specific peptide inhibitor of calcineurin in vitro as well as cyclosporin A, a cell-permeant inhibitor of calcineurin, in intact cells. Our results suggest that a single substrate might mediate opposing actions of PKC and calcineurin in the regulation of Abeta formation.
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PMID:Amyloid beta peptide formation in cell-free preparations. Regulation by protein kinase C, calmodulin, and calcineurin. 879 34

Phosphorylation of the microtubule-associated protein tau regulates its binding to microtubules; highly phosphorylated tau is also a prime component of paired helical filaments (PHFs) of Alzheimer's disease (AD). Tau from freshly biopsied human, monkey, and rat brain share similar electrophoretic mobility patterns and overlapping phosphorylated epitopes when compared to AD tau isolated from AD brain. We compared the microtubule reassembly competence of fresh isolates of phosphorylated tau to that of maximally dephosphorylated tau and tau from AD brain. A rapid procedure was developed which permitted the enrichment of phosphorylated and dephosphorylated tau from human biopsies in the absence of protein kinase and phosphatase activity. Microtubule assembly assays, using a spectrophotometric measure and purified bovine brain tubulin, were used to correlate assembly competence with states of tau electrophoretic mobility. Maximally dephosphorylated human biopsy-derived tau and monkey tau were assembly competent; tau from AD brain was virtually unable to direct microtubule assembly. Unmodified, biopsy-derived tau from non-AD brain was intermediate in assembly competence relative to AD tau and dephosphorylated tau. Several lines of evidence were used to correlate phosphorylation states of tau with microtubule assembly. First, in vitro dephosphorylation of human biopsy-derived tau with either PP2A or PP2B alone or in combination led to increasing assembly competence as the electrophoretic mobility of tau increased. Second, addition of the protein phosphatase inhibitor okadaic acid (10 microM) to brain-slice preparations slowed electrophoretic mobility of tau and decreased binding competence. We suggest that tau derived from freshly-biopsied brain exists in a range of phosphorylated states, and that dephosphorylation by PP2A and/or PP2B increases microtubule assembly competence.
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PMID:Microtubule assembly competence analysis of freshly-biopsied human tau, dephosphorylated tau, and Alzheimer tau. 892 25


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