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)

Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.
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PMID:Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay. 793 90

Hormone sensitive lipase (HSL) is an enzyme of relatively broad specificity, having the ability to hydrolyze tri-, di- and mono-acylglycerols as well as cholesterol esters and small water-soluble substrates. This broad specificity allows HSL to perform a variety of functions in several tissues. A key feature of HSL is its ability to be activated via phosphorylation by cyclic AMP-dependent protein kinase. In addition it is phosphorylated at a second site by several kinases, notably AMP-activated protein kinase. Phosphorylation of this site apparently plays a role in rendering the enzyme hormone-insensitive, in that prior phosphorylation at site 2 prevents phosphorylation and activation at site 1 by cyclic AMP-dependent protein kinase. Investigation of the protein phosphatases responsible for dephosphorylation of these sites has indicated that phosphatase 2A plays a predominant role but also that protein phosphatase 2C is a significant phosphatase targeted against both phosphorylation sites. Evidence indicates that HSL has at least three functional domains which contain (a) the phosphorylation sites which control activity, (b) the active site responsible for the catalytic activity and (c) a lipid binding site responsible for anchoring the lipase at the water-lipid interface. Using limited proteolytic studies we have found that it is possible to cleave HSL into several fragments including a stable domain of M(r) approximately 17.6 kDa which contains the active site serine residue. Digestion under similar conditions also generates a stable domain of M(r) approximately 11.5 kDa containing both phosphorylation sites. Furthermore, under appropriate conditions it is possible to digest HSL and retain activity against water-soluble substrates but with the concomitant loss of activity against triacylglycerol, implying that a lipid binding domain is lost during this procedure. HSL is responsible for the neutral cholesterol esterase activity in macrophages and it may play a role in the accumulation of cholesterol esters which occur during the development of foam cells. HSL activity is reduced in macrophage foam cells, at least partly due to increased activity of a cytosolic HSL inhibitor protein. A finding unexplained for many years has been that, although lipolysis can be stimulated 50-100-fold in adipocytes by lipolytic hormones, HSL can apparently only be activated 2-3-fold via phosphorylation in vitro by cyclic AMP-dependent protein kinase. One possibility to explain this discrepancy is that an additional anchoring protein is missing from the in vitro system and indirect evidence is now accumulating for such a protein.
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PMID:The multifunctional role of hormone-sensitive lipase in lipid metabolism. 794 81

A soluble protein phosphatase from the promastigote form of the parasitic protozoan Leishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42,000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg2+ ions were essential for enzyme activity; among other metal ions Mn2+ can replace Mg2+ to a certain extent whereas Ca2+, Co2+ and Zn2+ could not substitute for Mg2+. The pH optimum of the enzyme was 6.5-7.5 and the temperature optimum 37 degrees C. The apparent Km for phosphohistone was 7.14 microM. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest that L. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.
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PMID:Partial purification and characterization of a soluble protein phosphatase from Leishmania donovani promastigotes. 859 23

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr=32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1.
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PMID:Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons. 946 12

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.
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PMID:Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C. 1009 95

Using autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) as substrate, we now find that long-term potentian (LTP) induction and maintenance are also associated with a significant decrease in calyculin A-sensitive protein phosphatase (protein phosphatase 2A) activity, without changes in Mg2+-dependent protein phosphatase (protein phosphatase 2C) activity. This decrease in protein phosphatase 2A activity was prevented when LTP induction was inhibited by treatment with calmidazolium or D-2-amino-5-phosphonopentanoic acid. In addition, the application of high-frequency stimulation to 32P-labeled hippocampal slices resulted in increases in the phosphorylation of a 55-kDa protein immunoprecipitated with anti-phosphatase 2A antibodies. Use of a specific antibody revealed that the 55-kDa protein is the B'alpha subunit of protein phosphatase 2A. Following purification of brain protein phosphatase 2A, the B'alpha subunit was phosphorylated by CaM kinase II, an event that led to the reduction of protein phosphatase 2A activity. These results suggest that the decreased activity in protein phosphatase 2A following LTP induction contributes to the maintenance of constitutively active CaM kinase II and to the long-lasting increase in phosphorylation of synaptic components implicated in LTP.
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PMID:Decreased protein phosphatase 2A activity in hippocampal long-term potentiation. 1064 34

The family of the PII signal transduction proteins contains the most highly conserved signaling proteins in nature. The cyanobacterial PII-homologue transmits signals of the cellular nitrogen status and carbon status through phosphorylation of a seryl-residue. To identify the enzyme responsible for dephosphorylation of the phosphorylated PII protein in Synechocystis PCC 6803, prospective phosphatase encoding genes were inactivated by targeted insertion of kanamycin resistance cassettes. Disruption of ORF sll1771 generates a mutant unable to dephosphorylate PII under various experimental conditions. On the basis of conserved signature motifs, the sll1771 product (termed PphA) is a member of the protein phosphatase 2C (PP2C) superfamily, which is characterized by Mg(2+)/Mn(2+)-dependent catalytic activity. Biochemical analysis of overexpressed and purified PphA confirms its PP2C-type enzymatic properties and demonstrated its reactivity toward the phosphorylated PII protein. Thus, PphA is the first protein phosphatase in Synechocystis PCC 6803 for which the physiological substrate and function is known.
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PMID:A PP2C-type phosphatase dephosphorylates the PII signaling protein in the cyanobacterium Synechocystis PCC 6803. 1168 19

The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.
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PMID:Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C. 1169 56

Natriuretic peptide receptor (NPR)-A is the primary signaling receptor for atrial natriuretic peptide and brain natriuretic peptide. Ligand binding to NPR-A rapidly activates its guanylyl cyclase domain, but its rate of cGMP synthesis declines with time. This waning of activity is called homologous desensitization and is mediated in part by receptor dephosphorylation. Here, we characterize two distinct NPR-A phosphatase activities. The serine/threonine protein phosphatase inhibitor, microcystin, inhibited the desensitization of NPR-A in membrane guanylyl cyclase assays in the absence of magnesium. EDTA also inhibited the desensitization, whereas MgCl(2) stimulated the desensitization. Because the effects of microcystin and EDTA were additive, and microcystin did not block the magnesium-dependent desensitization, the targets for these agents appear to be distinct. Incubation of membranes at 37 degrees C stimulated the dephosphorylation of NPR-A, and microcystin blocked the temperature-dependent dephosphorylation. The addition of MgCl(2) or MnCl(2), but not CaCl(2), further stimulated the dephosphorylation of NPR-A, and microcystin failed to inhibit this process. The desensitization required changes in the phosphorylation state of NPR-A because the guanylyl cyclase activity of a receptor variant containing glutamate substitutions at all six phosphorylation sites was unaffected by MgCl(2), EDTA, or microcystin. Together, these data indicate that NPR-A is regulated by two distinct phosphatases, possibly including a member of the protein phosphatase 2C family. Finally, we observed that the desensitization of NPR-A in membranes from mouse kidneys and NIH3T3 cells was increased by prior exposure to atrial natriuretic peptide, suggesting that hormone binding enhances receptor dephosphorylation.
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PMID:The atrial natriuretic peptide receptor (NPR-A/GC-A) is dephosphorylated by distinct microcystin-sensitive and magnesium-dependent protein phosphatases. 1182 94

ABI1, a protein phosphatase 2C, is a key component of signal transduction in Arabidopsis. It regulates diverse responses to the phytohormone abscisic acid (ABA) such as stomatal closure, seed dormancy and inhibition of vegetative growth. By analysing proteins capable of interacting with ABI1, we have identified the homeodomain protein ATHB6 as a regulator of the ABA signal pathway. Critical for interaction between ATHB6 and ABI1 is an intact protein phosphatase domain and the N-terminal domain of ATHB6 containing the DNA-binding site. ATHB6 recognizes a cis-element present in its promoter, which encompasses the core motif (CAATTATTA) that mediated ATHB6- and ABA-dependent gene expression in protoplasts. In addition, transgenic plants containing a luciferase gene controlled by the ATHB6 promoter documented a strong ABA-inducible expression of the reporter which was abrogated in the ABA-insensitive abi1 mutant. Arabidopsis plants with constitutive expression of the transcriptional regulator revealed ABA insensitivity in a subset of ABI1-dependent responses. Thus, the homeodomain protein ATHB6 seems to represent a negative regulator of the ABA signal pathway and to act downstream of ABI1.
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PMID:Homeodomain protein ATHB6 is a target of the protein phosphatase ABI1 and regulates hormone responses in Arabidopsis. 1206 16


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