Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The skeletal muscle glycogen-binding subunit (GM) of protein phosphatase-1 (PP1) is the founding member of a family of proteins that tether the PP1 catalytic subunit (PP1C) to glycogen and promote the dephosphorylation of glycogen synthase. A hydrophobic sequence (called here the VFV motif) is conserved among GM, the liver subunit GL, and the widely expressed subunits, PTG, R5 and U5. This study analyzed the role of this VFV motif in binding to glycogen and PP1C. Glutathione S-transferase (GST) fusions with the N-terminal domain of GM (GST-GM(1-240)) and with the full length R5 protein (GST-R5) both bound to glycogen in a co-sedimentation assay. In contrast, GST itself did not bind to glycogen. A single residue substitution in GST-GM(1-240), F155A, reduced glycogen binding by 40%. Double residue substitutions V150A/F155A and F155A/V159A resulted in greater reductions (60-70%) in glycogen binding, showing these hydrophobic residues influenced the protein-glycogen interaction. The wild type and V150A/ F155A fusion proteins were digested by trypsin into the same sized fragments at the same rate. Furthermore, the wild type and mutated GST-GM proteins as well as GST-R5 bound equivalent amounts of PP1C, in either pull-down or far-Western assays. These results demonstrated retention of overall tertiary structure by the mutated fusion proteins, and indicated that glycogen and PP1C binding are independent of one another. A 68 residue segment of R5 encompassing the VFV motif was sufficient to produce glycogen binding when fused to GST. This motif, that is in bacterial and fungal starch metabolizing enzymes, probably has been conserved during evolution as a functional domain for binding glycogen and starch.
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PMID:A conserved domain for glycogen binding in protein phosphatase-1 targeting subunits. 984 3

Inside-out patch recordings from rat acutely dissociated cerebral cortical neurons revealed time and voltage-dependent activity of a large-conductance calcium-activated potassium channel. Channel activity inactivated within minutes following a depolarizing voltage step, and was recovered from inactivation by membrane hyperpolarization. Inactivation rate was not influenced by internal calcium or membrane voltage; however, reducing channel activity with intracellular calcium destabilized inactivation. Channel inactivation was abolished by intracellular trypsin treatment, suggesting that an associated inactivating particle was responsible for inactivation. Application of alkaline phosphatase to the internal aspect of the patch membrane increased channel activity and abolished channel inactivation, without affecting its voltage and calcium dependence. Internal application of Mg-ATP, but not Mg-5'-adenylylamidodiphosphate, retarded recovery of channel activity from inactivation, whereas internal application of protein phosphatase-1alpha enhanced recovery from inactivation. The abolition of channel inactivation by alkaline phosphatase was prevented by prior internal tetraethylammonium treatment, indicating that the alkaline phosphatase site is closely associated with the channel pore. These results demonstrate that cortical large-conductance calcium-activated potassium channel inactivation is probably mediated by an endogenous, trypsin-sensitive, inactivation particle. This particle appears to inactivate the open channel and requires a critical phosphate group for stable block. The slow time-course of channel inactivation may have some pathophysiological significance in maintenance of epileptiform activity.
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PMID:Inactivation of large-conductance, calcium-activated potassium channels in rat cortical neurons. 1061 60

The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its beta1 and mu1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its beta1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated beta1 compared with phosphorylated mu1. Once on the membrane, the mu1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of mu1 (and mu2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.
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PMID:AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation. 1260 86

Type 1 protein phosphatase (PP1) is a negative regulator of cardiac function. However, studies on the status and regulation of sarcoplasmic reticulum (SR)-associated PP1 activity in failing hearts are limited. We studied PP1 activity and protein and mRNA expression of the catalytic subunit of PP1 (PP1C) and protein levels of PP1-specific inhibitors [inhibitor 1 (Inh-1) and inhibitor 2 (Inh-2)] in the left ventricular (LV) myocardium of 6 dogs with heart failure (HF; LV ejection fraction, 23 +/- 2%) and 6 normal dogs. In failing LV tissue, PP1 activity values (expressed as pmol 32P. min-1. mg of noncollagen protein-1) in the homogenate, crude membranes, cytosol, and purified SR were increased by 52, 54, 55, and 72%, respectively. Trypsin treatment released PP1 but not type 2A protein phosphatase from the SR. In the supernatant of trypsin-treated SR, PP1 activity was approximately 24% higher in failing hearts than in normal control hearts. A similar increase in protein expression of PP1C was observed in the nontrypsinized SR. Heat-denatured phosphorylated SR inhibited PP1 activity by 30%, which suggests the presence of Inh-1 or -2 or both in the SR. With the use of a specific antibody, both Inh-1 and -2 proteins were found in the SR; the former was decreased by 56% in the failing SR, whereas the latter did not change. These results suggest that protein phosphatase activity bound to the SR is increased and is predominantly type 1. Increased SR-associated PP1 activity in failing hearts appears to be due partly to increased expression of PP1C and partly to reduced levels of Inh-1 but not Inh-2 protein. Thus inhibition of PP1 activity in the SR appears to be a potential therapeutic target for improving LV function in failing hearts, because it may lead to increased SR Ca2+ uptake, which is impaired in failing hearts.
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PMID:Cardiac SR-coupled PP1 activity and expression are increased and inhibitor 1 protein expression is decreased in failing hearts. 1461 11

The protease-activated receptor-2 (PAR-2) has been implicated in airway inflammation. Here, we examined the interaction between PAR-2 and lipopolysaccharide (LPS), a major proinflammatory factor, using cultured guinea pig tracheal epithelial cells. In fura2-loaded cells, LPS (1 microg/ml) transiently increased intracellular Ca(2+) concentrations ([Ca(2+)]i), this effect being abolished by a Ca(2+) channel blocker, verapamil, and Ca(2+) removal. Prestimulation of PAR-2 with trypsin (0.1-1 U/ml) or an agonist peptide (SLIGRL-NH(2), 1 microM) for 60 min inhibited the LPS-induced [Ca(2+)]i increase. Such an inhibitory effect of trypsin was abolished by inhibitors of protein kinase C (PKC), chelerythrine and staurosporine. A PKC activator, phorbol 12,13-dibutylate, also reduced the LPS response. Trypsin also inhibited a transient increase in [Ca(2+)]i caused by a Ca(2+) channel opener, Bay K 8644. When the trypsin-pretreated cells were incubated in normal buffer for 10-60 min before LPS exposure, the effect of trypsin on the Ca(2+) response to LPS diminished in a time-dependent manner. Such a recovery was slowed by incubation with a protein phosphatase inhibitor, okadaic acid. Further, trypsin induced sustained activations of PKCalpha and -epsilon. Thus, PAR-2 stimulation reduced the epithelial cell response to LPS, probably through the inactivation of Ca(2+) channels via PKC-mediated phosphorylation.
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PMID:Protease-activated receptor-2-mediated inhibition for Ca2+ response to lipopolysaccharide in Guinea pig tracheal epithelial cells. 1475 55

1 In this study, we examined the role of Ca2+ in linking proteinase-activated receptor-2 (PAR2) to the nuclear factor kappa B (NFkappaB) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2-mediated NFkappaB luciferase reporter activity and NFkappaB DNA-binding activity was reduced by preincubation with BAPTA-AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKKalpha and IKKbeta, was also inhibited following pretreatment with BAPTA-AM. 3 BAPTA/AM also prevented PAR2-mediated IKKalpha activation in cultured primary human keratinocytes. 4 The effect of BAPTA-AM was also selective for the IKK/NFkappaB signalling axis; PAR2 coupling to ERK, or p38 MAP kinase was unaffected. 5 Pharmacological inhibition of the Ca2+-dependent regulatory protein calcineurin did not inhibit trypsin-stimulated IKK activity or NFkappaB-DNA binding; however, inhibition of Ca2+-dependent protein kinase C isoforms or InsP3 formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFkappaB-DNA binding. 6 Mutation of PAR2 within the C-terminal to produce a mutant receptor, which does not couple to Ca2+ signalling, but is able to activate ERK, abrogated NFkappaB-DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFkappaB transcriptional activation.
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PMID:The role of intracellular Ca2+ in the regulation of proteinase-activated receptor-2 mediated nuclear factor kappa B signalling in keratinocytes. 1582 58

It is well known that the activity of calcineurin (CaN) could be modulated by several transitional metal ions. In the present work, the effects of a calcium analog, lanthanum ion (La(3+)), on the activity of CaN were studied. It was found that La(3+) exerted multiple effects on CaN activity. La(3+) could stimulate CaN in the absence of calmodulin (CaM); whereas at low concentrations of La(3+), there was a slight inhibition of activation of CaN in the presence of CaM. Competitive experiments and limited trypsin proteolysis confirmed that La(3+) did not act on the catalytic core of CaN, but exerted its effect through direct action on the CaN regulatory domain similar to Mg(2+). In activity titration and spot blotting studies, La(3+)-containing CaM complexes were less effective in stimulating CaN than Ca(2+) or Mn(2+)-containing CaM; however, the binding affinity of these metal-CaM complexes to CaN was similar. These effects of La(3+) on CaN activity are unique among metal ions and may provide clues to understand the biological effects of La(3+).
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PMID:La3+ stimulate the activity of calcineurin in two different ways. 1617 24

Using mouse hearts from Swiss Webster mice, calcineurin was immunoprecipitated using commercially available anti-calcineurin antibody and the resulting complex analyzed by using sodium dodecyl sulfate-gel electrophoresis with silver staining. Distinct proteins were observed and subjected to in situ trypsin digestion followed by extraction of the resulting peptides. Peptides from each protein band were loaded onto a target for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and analyzed. The resulting peptide mass spectrum was compared with the Mascot and Protein Prospector databases and resulted in the specific identification of heart mitochondrial proteins, specifically Mn-superoxide dismutase (SOD), aconitase (ACN), and malate dehydrogenase (MDH). Each of the three mitochondrial enzymes was identified with approximately 15-25% sequence coverage and all with statistical significance (P < 0.05) according to the Mascot database search engine. Tandem mass spectrometry analysis of the peptide fragmentation spectra confirmed the identification of these protein partners and also yielded the identification of mitochondrial isocitrate dehydrogenase (ICDH) as another protein in the immunoprecipitated complex. Using antibody preparations against Mn-SOD, ACN, and ICDH showed the presence of calcineurin and each of the three proteins in the immunoprecipitated complex by Western slot blotting. The activity of ACN, but not MDH or ICDH, was enhanced after incubation with calcineurin indicating one possible regulatory function for the complex. The mitochondrial forms of Mn-SOD, ACN, MDH, and ICDH were identified as partner proteins of calcineurin with all the proteins present in a single multiprotein complex.
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PMID:Association of calcineurin with mitochondrial proteins. 1663 48

Calcium is a second messenger for many signaling pathways in B cells, but its role as a receptor ligand has not been well characterized. However, pulses of free calcium were found to cause the rapid release of internal calcium stores in normal human B cells. This response appeared to be mediated by a cell surface protein with receptor properties as it could be blocked by pretreatment with trypsin and with kinase and phospholipase Cgamma inhibitors. The calcium receptor on B cells was not the conventional calcium-sensing receptor (CaSR) since B cells did not express CaSR and calcium-induced responses could not be blocked by specific CaSR inhibitors. B-cell responses to extracellular calcium activated phosphoinositide-3 kinase/AKT, calcineurin, extracellular signal regulated kinase, p38 mitogen-activated protein kinase, protein kinase C, Ca(2+)/calmodulin kinase II, and nuclear factor-kappaB signaling pathways, and resulted in transcription of the early response gene, CD83. This extracellular calcium sensor enhanced B-cell responses to Toll-like receptor, B-cell receptor, and cytokine receptor agonists. These findings suggest a means by which B cells prepare to engage in immune responses by responding to calcium fluctuations in their environment.
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PMID:Extracellular calcium sensing promotes human B-cell activation and function. 1772 42

Pathologic responses arising from the pancreatic acinar cell appear to have a central role in initiating acute pancreatitis. Environmental factors that sensitize the acinar cell to harmful stimuli likely have a critical role in many forms of pancreatitis, including that induced by alcohol abuse. Activation of zymogens within the acinar cell and an inhibition of secretion are critical, but poorly understood, early pancreatitis events. While there is firm evidence relating trypsinogen activation to pancreatitis, the importance of other zymogens has been less studied. Preliminary studies suggest that trypsin may be activated by mechanisms that are distinct from other zymogens. Further, unlike the small intestine, it may not catalyze the activation of other zymogens. These features could affect strategies aimed at inhibiting proteases to treat pancreatitis. Specific intracellular signals are required to activate pancreatitis pathways in the acinar cell. The most important is calcium. Recent studies have suggested that calcium release through specific calcium channels in the endoplasmic reticulum is the means by which pathological elevations in cytosolic calcium occur. Although the targets of abnormal calcium signaling are unknown, calcineurin, a calcium-dependent phosphatase, may serve such a role. Finally, recent work suggests that an acute acid load might sensitize the acinar cell to pancreatitis responses. Therapies aimed at preventing or reversing the effects of an acid load on the pancreas may be important for treatment.
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PMID:The acinar cell and early pancreatitis responses. 1989 90


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