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)

Calponin is a basic, approximately 34,000 M(r), smooth muscle-specific protein which is developmentally expressed in up to four isoforms. Calponin binds very strongly to actin in a Ca(2+)-independent manner and is localized to the thin filaments in smooth muscle, where it is present at a stoichiometry of 1 mol calponin/7 mol actin. The interaction of calponin with actin inhibits the actomyosin MgATPase (cross-bridge cycling rate) without affecting myosin phosphorylation. The calponin-actin interaction is blocked and calponin-mediated inhibition of the actomyosin MgATPase is reversed upon phosphorylation of calponin by either PKC or CaM kinase II; these properties are restored upon dephosphorylation of calponin by a type 2A protein phosphatase. Consistent with these in vitro findings, calponin is phosphorylated in intact smooth muscle in response to contractile stimuli. The increasing body of evidence, both in vitro and in vivo, strongly supports calponin phosphorylation-dephosphorylation as a thin filament-linked regulatory system in smooth muscle.
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PMID:Calponin: thin filament-linked regulation of smooth muscle contraction. 813 72

The phosphorylation of one receptor that occurs as a result of the stimulation of a different receptor on a cell is a common mechanism for heterologous regulation or "cross-talk," which has been implicated in desensitization. In this work, we focus on the mechanisms of phosphorylation of the rat pancreatic acinar cell cholecystokinin (CCK) receptor that occur upon stimulation of this cell by various agonists. Phosphorylation was allowed to occur in dispersed intact acinar cells in response to the experimental manipulation, and the phosphoreceptor was subsequently purified and quantified as an indication of response. Agonists such as vasoactive intestinal polypeptide and secretin, which act via activation of adenylate cyclase, had no effect on CCK receptor phosphorylation, whereas carbamylcholine and bombesin stimulated increased phosphorylation of the CCK receptor. Because these agents would be expected to activate protein kinase C (PKC) as well as a number of calcium-sensitive kinases and phosphatases, these activities were further dissociated by using more direct activators and inhibitors acting intracellularly. Manipulation of calcium independent of PKC by using a calcium ionophore, inhibition of calcium/calmodulin-dependent kinase II, and inhibition of calcium-dependent protein phosphatase type 2B had no effect on the state of CCK receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanisms of heterologous agonist-stimulated phosphorylation of cholecystokinin receptor. 817 63

The Ca(2+)-dependent protease, calpain II, isolated from vascular smooth muscle was found to be a substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in vitro. Phosphorylation was dependent upon prior autolysis of the regulatory subunit of calpain II. The stoichiometry of phosphorylation of native, unautolyzed calpain II was 0.02 +/- 0.01 mol PO4/mol enzyme while for autolyzed calpain, the stoichiometry was 1.04 +/- 0.15 mol PO4/mol enzyme. All phosphate was incorporated into the 76 kDa catalytic subunit of calpain II. A single serine residue in domain III of the catalytic subunit was identified as the phosphate acceptor: RGS*TAGGCR. Phosphorylation doubled enzyme activity measured both as proteolysis of an exogenous substrate (alpha-casein) as well as by intermolecular catalytic subunit autolysis. The effects of phosphorylation could be reversed by dephosphorylation using a type IIA phosphoprotein phosphatase. These results demonstrate that calpain II possesses a latent CaM kinase II phosphorylation site that is unmasked by autolysis.
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PMID:Identification of a latent Ca2+/calmodulin dependent protein kinase II phosphorylation site in vascular calpain II. 818 34

We have characterized Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV), expressed using the baculovirus/Sf9 cell system, to assess its potential role in Ca2+-dependent transcriptional regulation. CaM kinase IV was strongly inhibited in vitro by KN-62, a specific CaM kinase inhibitor which suppresses Ca2+-dependent transcription of several genes, so we tested whether CaM kinase IV could stimulate transcription. Co-transfection of COS-1 cells by cDNA for CaM kinase IV gave 3-fold stimulation of a reporter gene expression, whereas co-transfection with CaM kinase II gave no transcriptional stimulation. Since this transcriptional response was mediated by phosphorylation of cAMP responsive element-binding protein (CREB), we determined the kinetics and site specificities of CaM kinases IV and II for phosphorylating CREB in vitro. CaM kinases IV and II and cAMP kinase (protein kinase A) all had similar Km values for CREB (1-5 microns), but the Vmax of CaM kinase IV was 40-fold lower than those of CaM kinase II and protein kinase A. Although all three kinases phosphorylated Ser133 in CREB, CaM kinase II also gave equal phosphorylation of a second site which was not Ser98. The two CREB phosphorylation sites were separately 32P-labeled, and the abilities of protein phosphatases 1, 2A, and 2B (calcineurin) to dephosphorylate them were tested. Our results show that all three phosphatases could dephosphorylate both sites, and calcineurin was a stronger catalyst for dephosphorylating site 1 (Ser133) than for site 2. These results indicate that CaM kinase IV may be important in Ca2+-dependent transcriptional regulation through phosphorylation of Ser133 in CREB. The fact that CaM kinase II phosphorylates another site in addition to Ser133 in CREB raises the possibility that this second phosphorylation site may account for the suppressed phosphorylation site may account for the suppressed ability of CaM kinase II to enhance transcription through the CRE/CREB system. In addition multiple protein phosphatases, including calcineurin, may exert a modulatory effect on transcription depending on which site they dephosphorylate.
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PMID:Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation. 819 96

Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.
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PMID:Calponin phosphorylation in vitro and in intact muscle. 828 82

A permeable cell system in which Ca2+ release can be evoked by inositol 1,4,5-trisphosphate (IP3) or agonist stimulation was used to study the regulation of Ca2+ release by Ca2+ itself. At low concentrations, Ca2+ activated IP3-mediated Ca2+ release (IMCR) with half-maximal effect at about 15 nM. At high concentrations, Ca2+ inhibited IMCR giving rise to a biphasic [Ca2+] dependence of IMCR. The activation of IMCR by Ca2+ appears to be mediated by a kinase, probably the Ca(2+)-and calmodulin-dependent protein kinase (CaMKII). Thus, the activation required MgATP, completely blocked at 0 degrees C, required Ca2+, and was inhibited by the CaMKII inhibitors KT5926 and KN62. The inhibition of IMCR seems to be mediated by a protein phosphatase, probably the Ca(2+)-dependent protein phosphatase 2B. Hence, the inhibition required Ca2+, was prevented by the general protein phosphatase inhibitor pyrophosphate and by the immunosuppressants cyclosporin A and FK506, but not by okadaic acid or VO4(2-), and was modified by chelating agents such as EGTA. Stimulation with agonists modified the activities of the kinase and phosphatase to make the release independent of [Ca2+]. This appears to be due to an increase in the apparent affinity for Ca2+ in stimulating IMCR and inhibition of the phosphatase. We suggest that agonist-dependent modification of the kinase/phosphatase activity ratio can be the biochemical pathway responsible for regulation of Ca2+ release and in turn [Ca2+]i oscillations.
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PMID:Ca(2+)-dependent kinase and phosphatase control inositol 1,4,5-trisphosphate-mediated Ca2+ release. Modification by agonist stimulation. 838 79

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.
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PMID:Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase. 839 39

Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of cAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.
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PMID:Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors. 839 87

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

The preproendothelin-1 (preproET-1) gene is induced by thrombin after phosphorylation of nonreceptor protein tyrosine kinase pathways. This study investigated the contribution of Ca2+/calmodulin-dependent intracellular signaling cascades to this pathway and measured ET-1 mRNA levels by Northern blot analysis in human endothelial cells. Increased intracellular Ca2+ levels in response to Ca2+ ionophore or Ca2+ ATPase inhibitors tert-butylhydroquinone and thapsigargin mimicked thrombin actions on ET-1 mRNA induction. Thrombin-mediated activation of ET-1 mRNA was reduced by specific calmodulin antagonists W7 or calmidazolium and after inhibition of CaM kinase II by KN-62. Inhibition of calcium/calmodulin-dependent phosphatase calcineurin by cyclosporin A, however, stimulated ET-1 mRNA in human endothelial cells. Phosphotyrosine immunoblot assays show that calcium/calmodulin-dependent signaling pathways precede thrombin-induced tyrosine phosphorylation, and that the calcium/calmodulin-dependent phosphatase calcineurin also exerts its effects via activation of protein tyrosine kinases. These observations demonstrate that thrombin stimulates the preproET-1 gene in human endothelial cells through calcium-dependent activation of CaM kinase and protein tyrosine kinases, and that calcineurin may also participate in regulation of the prepro ET-1 gene.
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PMID:Thrombin-mediated ET-1 gene regulation involves CaM kinases and calcineurin in human endothelial cells. 858 30


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