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)

We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.
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PMID:Regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II and calcineurin in the hippocampus of rat brain after transient forebrain ischemia. 131 54

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

Characteristics of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the cytosol and in the postsynaptic densities (PSD) of rat brain were investigated. Several proteins were surveyed for their abilities to serve as a substrate for non-autophosphorylated and autophosphorylated CaM kinase IIs from the cytosol and PSD. The tested substrates were separated into two groups. Autophosphorylation of the kinase slightly decreased or did not change its activities towards substrates of the first group: myosin light chain of chicken gizzard, synapsin I, tau factor and microtubule-associated protein 2. In contrast, autophosphorylation of the enzyme increased its activities towards substrates of the second group: syntide-2, histone H1, calcineurin and myelin basic protein. The Ca2+/calmodulin-independent kinase activity increased by autophosphorylation with any of substrates tested. Similar results were obtained with the cytosolic and PSD CaM kinase II. Trifluoperazine and mastoparan, calmodulin binding antagonists, inhibited the activity of the non-autophosphorylated CaM kinase II, but had no effect or only a slight inhibitory effect on the activity of the autophosphorylated CaM kinase II, indicating that the autophosphorylated kinase has no requirement for calmodulin for Ca(2+)-dependent activity and/or a higher affinity for calmodulin The results suggest that the autophosphorylation of CaM kinase II is a subtle mechanism for regulating the interaction between the enzyme and substrate.
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PMID:Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II: effects on interaction between enzyme and substrate. 164 40

A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release.
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PMID:Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr. 190 12

When the stimuli by nerve impulses, neurotransmitters, hormones, peptides and growth factors are administered to the neurons, one of the responses of the nerve cells is the enhancement of Ca2+ influx and/or the release of Ca2+ from the intracellular storage site. Ca2+ may be related to several types of neuronal functions such as biosynthesis of neurotransmitters, stimulus-secretion coupling of neurotransmitters and hormones, microtubule assembly-disassembly cycle and many metabolic reactions. Although the precise molecular mechanism mediating the actions of Ca2+ in the brain remains to be elucidated, accumulating evidence suggests that the actions of Ca2+ are mediated through Ca2(+)-binding proteins. The role of troponin C, a Ca2(+)-binding protein, was extensively studied in the skeletal muscle first. Subsequently calmodulin, a ubiquitous Ca2(+)-binding protein, was found to be widely distributed in many tissues and to be involved in a variety of Ca2(+)-mediated cellular processes. In an attempt to elucidate Ca2+ actions in the central nervous system, we have been studying Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase). These enzymes have many common substrates and, therefore, may be involved in the neuronal functions via phosphorylation and dephosphorylation of specific proteins.
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PMID:[A study on the role and mechanism of intracellular Ca2+ in the central nervous system]. 217 89

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.
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PMID:Expression of calmodulin-dependent phosphodiesterase, calmodulin-dependent protein phosphatase, and other calmodulin-binding proteins in human SMS-KCNR neuroblastoma cells. 254 Feb 70

Recent studies indicate multiple mechanisms are involved in Ca2+ stimulation of gene expression. We have used cell-permeable, specific inhibitors of calmodulin-dependent protein kinases (CaM kinases) and phosphatase (calcineurin) to investigate the involvement of these enzymes in transcriptional regulation of three immediate early genes in PC12 cells stimulated with A23187 or KCl. Preincubation of PC12 cells with the CaM kinase inhibitor KN-62 blocked autophosphorylation of CaM kinase II in response to stimulation by the Ca2+ ionophore A23187. KN-62 treatment also resulted in a 60-70% inhibition of Ca(2+)-dependent transcription of c-fos, NGFI-A (zif 268), and NGFI-B (nur 77) as assessed by either Northern or nuclear run-on analyses. Preincubation with the calcineurin inhibitors FK-506 or cyclosporin A strongly enhanced expression of NGFI-A and blocked transcription of NGFI-B, but it had no significant effect on Ca(2+)-stimulated transcription of c-fos. Both FK-506 and KN-62 were specific for Ca(2+)-stimulated transcription as neither effected transcription in response to forskolin or phorbol ester (12-O-tetradecanoylphorbol-13-acetate) treatment. This is the first report of CaM kinase and calcineurin involvement in transcriptional regulation of NGFI-A and NGFI-B. Activation of CaM kinases and calcineurin, in response to elevated intracellular Ca2+, would exert antagonistic effects on transcription of NGFI-A. Since inhibition of either the kinase or phosphatase decreased transcription of NGFI-B by 60-90%, this suggests that each enzyme is necessary but not sufficient for Ca2+ stimulation. These results indicate that CaM kinases and calcineurin can mediate broad and complex regulation of Ca(2+)-stimulated gene expression.
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PMID:Roles of calmodulin-dependent protein kinases and phosphatase in calcium-dependent transcription of immediate early genes. 752 Apr 33

Calponin is a smooth muscle-specific, thin filament-associated protein which has been implicated in the regulation of contraction via its interaction with actin and inhibition of the cross-bridge cycling rate. Calponin is phosphorylated by protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), primarily at S175, with loss of actin binding and inhibition of the actin-activated myosin MgATPase. We previously isolated calponin phosphatase from chicken gizzard smooth muscle and identified it as a type 2A protein phosphatase [Winder et al. (1992) Biochem. J. 286, 197-203]. The methods used to detect phosphatase activity in that study would additionally have detected type 1 and 2C phosphatases, but not type 2B phosphatase (Ca2+/CaM-dependent phosphatase or calcineurin). We have, therefore, examined the expression of type 2B phosphatase in smooth muscle and its ability to dephosphorylate calponin. Western blotting with polyclonal antibodies to the brain enzyme revealed the expression of type 2B phosphatase in chicken gizzard, and immunofluorescence microscopy confirmed the presence of the phosphatase in isolated smooth muscle cells (rabbit and toad stomach). The purified brain phosphatase dephosphorylated calponin (phosphorylated by PKC or CaM kinase II) in a Ca2+/CaM-dependent manner. Dephosphorylation by calcineurin restored actin-binding and actin-activated myosin MgATPase inhibition which had been reduced by PKC-catalyzed phosphorylation. We conclude that calponin dephosphorylation may be catalyzed not only by type 2A phosphatase but also by type 2B phosphatase, raising the possibility that both phosphorylation and dephosphorylation of calponin could be regulated by Ca2+/CaM.
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PMID:Dephosphorylation of calponin by type 2B protein phosphatase. 761 14

Glucocorticoid hormones (GC) have profound effects on the development and homeostasis of the immune system. In this communication we present evidence that GC regulate Ca(2+)-mediated pathways of T cell activation by a mechanism that involves abrogation of the autophosphorylation of the multifunctional Ca2+/calmodulin kinase (CaM kinase II) and induction of protein phosphatase activity. Primary human T cells were stimulated with the combination of ionomycin and phorbol ester in the presence or absence of dexamethasone (Dex) (10(-6)-10(-12) M). Stimulation of T cells resulted in a rapid activation of CaM kinase II and protein kinase C (PKC) activity as determined by the phosphorylation of synthetic peptide substrates recognized by these enzymes. Dex inhibited the activity of CaM kinase II but not PKC activity in a dose-dependent fashion (minimum effective dose 10(-10) M). Stimulation of 32P-labeled T cells induced a rapid increase in the phosphorylation level of CaM kinase II which was inhibited by Dex. The inhibitory effect of Dex on this enzyme was fully reversed in the presence of the phosphatase inhibitor okadaic acid (250 nM) or RU 486, a glucocorticoid antagonist. These results suggest that GC inhibit the activation of CaM kinase during T cell activation through a mechanism that involves both the GC receptor and protein phosphatases 2A and/or 1. Inhibition of protein phosphorylation through the induction of protein phosphatase activity may represent a novel mechanism for the diverse effects of GC on eukaryotic cells.
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PMID:Glucocorticoid-mediated regulation of protein phosphorylation in primary human T cells. Evidence for induction of phosphatase activity. 763 35

We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase. 772 17


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