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 developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.
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PMID:Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells. 358 24

Interactions of several divalent cations (Mn2+, Ca2+, Co2+, Sr2+, and Zn2+) with EGTA-inhibitable adenylate cyclase were investigated in washed membranes (particles) isolated from the gray matter of rat cerebral cortex. The EGTA-inhibitable (called sensitive) enzyme activity was assayed in the presence of Triton X-100 since this detergent caused a marked increase (up to 20-fold) in the enzyme activity. The effects of various divalent metals (all added as chloride salt) indicated the presence of two distinct sites called site I and site II. At low concentrations (less than micromolar) Mn2+, Co2+, and Ca2+ increased (up to 10-fold) the enzyme activity to the same extent and appeared to act via binding to site I (high affinity site). The rank order of affinity was Mn2+ greater than or equal to Co2+ greater than Ca2+. Zn2+ showed the highest affinity and Sr2+ the lowest towards binding to site I; both these metals increased the enzyme activity to lesser extents than Mn2+, Co2+, or Ca2+. GTP was not required for the stimulation of this enzyme by low concentrations of Ca2+. The interaction of Mn2+ with site II (low affinity site) caused further increase in the enzyme activity, whereas Co2+, Ca2+, and Sr2+ were inhibitory at concentrations greater than 10 microM. Isolated fraction contained loosely and tightly associated pools of calmodulin. Myelin basic protein, but not calcineurin, inhibited the EGTA-sensitive adenylate cyclase activity. The EGTA-insensitive enzyme activity was increased by norepinephrine by mechanisms that depended on GTP and was inhibited by Ca2+. The stimulation of the EGTA-insensitive enzyme modulated the Mg2+ requirement such that Mg2+ binding to the low affinity site (site II) apparently occurred with higher affinity. The likely significance of these results is discussed with regard to (i) the presence of two classes of adenylate cyclase in rat cerebral cortex gray matter and (ii) the regulation of their activities by calmodulin-requiring and GTP-requiring mechanisms.
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PMID:EGTA-sensitive and -insensitive forms of particulate adenylate cyclase in rat cerebral cortex: regulation by divalent cations and GTP. 393 3

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.
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PMID:Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes. 625 May 98

Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.
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PMID:Phosphorylation of rat liver nuclear envelopes. I. Characterization of in vitro protein phosphorylation. 630 4

Branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of approximately 460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of approximately 230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations. BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 microM, respectively. These inhibitions were reversed completely by 2 mM Mg2+. GTP was replaceable by guanosine 5'-(beta, gamma-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 micrograms/ml. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 microM. These inhibitions were not reversed by 2 mM Mg2+. BCKDH phosphatase activity was stimulated 1.5- to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 micrograms/ml.
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PMID:Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney. 658 97

1. To clarify the nature of the inhibition of whole-cell inwardly rectifying K+ current (IK1) by isoprenaline (Iso) and its antagonism by acetylcholine (ACh), we studied the effects of Iso and ACh and their surrogates on single channel currents (iK1) carried by inwardly rectifying K+ channels in cell-attached and excised inside-out patches obtained from guinea-pig ventricular myocytes. 2. Bath application of Iso suppressed iK1 channel activity in cell-attached patches. This was inhibited by propranolol. Bath-applied forskolin or dibutyryl cAMP mimicked the effect of bath-applied Iso. 3. Exposure of the cytosolic face of inside-out patches to purified catalytic subunit of the cAMP-dependent protein kinase (PKA) also suppressed iK1 channel activity, mimicking the effect of bath-applied Iso on iK1 recorded from cell-attached patches. 4. When applied directly to cell-attached patches via the patch pipette solution, ACh antagonized Iso-induced (1 microM applied via the bath) suppression of iK1 channels. In contrast, bath-applied ACh (10 microM) partially antagonized the effect of low concentrations of Iso (e.g. < 50 nM) on iK1 channels in cell-attached patches but had no detectable effect when 1 microM or more Iso was used. 5. In myocytes pretreated with pertussis toxin (PTX), ACh failed to antagonize Iso-induced suppression of iK1 channels. When inside-out patches were used, bath-applied preactivated exogenous inhibitory G protein subunit, G1 alpha, antagonized the suppression of iK1 channels induced by bath-applied catalytic subunit of PKA (PKA-CS), suggesting that a PTX-sensitive G1 alpha mediates ACh-induced antagonism of Iso-induced suppression of iK1. 6. Neither GTP gamma S nor G1 alpha antagonized the suppression of iK1 produced by bath-applied PKA-CS in inside-out patches when okadaic acid was present in the bath. In addition, bath application of alkaline phosphatase also reactivated iK1 channels suppressed by PKA-CS. 7. Findings in guinea-pig ventricular myocytes suggest that iK1 can be suppressed by a PKA-mediated phosphorylation of the iK1 channel occurring in response to Iso-induced beta-adrenergic receptor activation and that ACh can antagonize the suppression by mechanisms that involve both intracellular and membrane-delimited pathways. The membrane-delimited pathway appears to involve M2-cholinergic receptors, their associated G protein, G1, and a protein phosphatase, all located in the sarcolemma in close proximity to the involved iK1 channels.
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PMID:Beta-adrenergic and cholinergic modulation of inward rectifier K+ channel function and phosphorylation in guinea-pig ventricle. 747 27

Intracellular recordings were obtained from myenteric AH neurons of guinea pig ileum in vitro. Slow excitatory synaptic responses associated with decreased potassium conductance (gK), inhibition of the spike afterhyperpolarization current (AHC), and increased chloride conductance (gCl) were mimicked by senktide, a neurokinin3 receptor agonist. Intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) decreased gK and increased gCl irreversibly after nerve stimulation or senktide application. Myenteric neurons in pertussis toxin (PTX)-treated tissues responded normally to senktide and nerve stimulation. Forskolin and phorbol 12,13-dibutyrate (PDBu) inhibited gK and the AHC but did not activate gCl. The AHC was not reduced by subthreshold concentrations of forskolin (10 nM) or PDBu (3 nM) alone but was inhibited by forskolin and PDBu applied together. Inhibitors of phospholipase C (D-609) or protein kinases (staurosporine) reduced slow synaptic and senktide responses. The protein phosphatase inhibitor, calyculin A, caused an inward current, a decrease in gK, and AHC inhibition but did not activate gCl. We conclude that slow excitatory synaptic responses are mediated by PTX-insensitive G proteins and activation of phospholipase C and protein kinases. Forskolin and PDBu activate pathways that inhibit gK. The mechanisms for activation of gCl are unknown.
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PMID:Signal-transduction pathways causing slow synaptic excitation in guinea pig myenteric AH neurons. 749 63

1. Exogenous arachidonic acid (AA) inhibits the protein phosphatase that dephosphorylates smooth muscle myosin, thus sensitizing the contractile response to Ca2+; it also inhibits voltage-gated Ca2+ channels in smooth muscle. The purpose of the present study was to determine whether endogenous AA is increased by agonists in a manner consistent with its role as a messenger regulating myosin phosphatase and Ca2+ channels. Both AA and diacylglycerol (DAG) were measured in [3H]AA-labelled intact and permeabilized (with staphylococcal alpha-toxin) rabbit femoral arteries stimulated with the alpha 1-adrenergic agonist phenylephrine (PE) (intact and permeabilized smooth muscles) or by guanosine-5'-O-(3-thiotriphosphate (GTP gamma S; permeabilized smooth muscles in which the [Ca2+] was maintained constant). Arachidonic acid mass was determined with gas chromatography and mass spectrometry (GC-MS). 2. In intact smooth muscle, PE increased both AA and DAG levels significantly, to 210 and 145% of baseline values, respectively. Another Ca2+-sensitizing agent, the thromboxane analogue U46619, caused a similar increase in AA and DAG levels in rabbit pulmonary artery. 3. In permeabilized smooth muscle at constant [Ca2+](pCa 6.5) GTP gamma S-induced AA and DAG release preceded force development and GTP gamma S (50 microM, 10 min) increased AA mass to 61-88 microM. 4. Phorbol-12,13-dibutyrate (PDBu), another Ca2+-sensitizing agent, also increased both AA and DAG levels in permeabilized smooth muscle at pCa 6.5, whereas the inactive analogue, 4 alpha-phorbol, did not have a Ca2+-sensitizing effect, nor did it increase AA and DAG levels. 5. In the virtual absence of Ca2+ (pCa > 8) GTP gamma S also increased AA and DAG levels by 3.5- and 1.6-fold, respectively. The effect of free Ca2+ itself on AA and DAG release was modest in the physiological range (pCa 7.0 to pCa 6.0), but pCa 4.5 caused an approximately 3- to 4-fold increase in AA and DAG levels, compared with the levels at pCa 8. In permeabilized ileum smooth muscle maintained at constant [Ca2+] (pCa 6.0), carbachol also significantly increased AA to 1.75 times its original value within 1 min of its application. 6. Our results are consistent with, although do not prove, the roles of AA and DAG as second and/or co-messenger(s) in smooth muscle, while the increases in AA and DAG levels induced by PDBu raise the possibility that they contribute to some of the cellular effects of phorbol esters.
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PMID:Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle. 756 27

1. Activation of human D2(s) dopamine receptors with quinpirole (10 nM) inhibits omega-conotoxin GVIa-sensitive, high-threshold calcium currents when expressed in differentiated NG108-15 cells (55% inhibition at +10 mV). This inhibition was made irreversible following intracellular dialysis with the non-hydrolysable guanosine triphosphate analogue GTP-gamma-S (100 microM), and was prevented by pretreatment with pertussis toxin (1 microgram ml-1 for 24 h). 2. Stimulation of protein kinase C with the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (100 microM), also attenuated the inhibition of the sustained calcium current but did not affect the receptor-mediated decrease in rate of current activation. Similarly, okadaic acid (100 nM), a protein phosphatase 1/2A inhibitor, selectively occluded the inhibition of the sustained current. 3. The depression of calcium currents by quinpirole (10 nM) was enhanced following intracellular dialysis with 100 microM cyclic adenosine monophosphate (cyclic AMP, 72.8 +/- 9.8% depression), but was not mimicked by the membrane permeant cyclic GMP analogue, Sp-8-bromoguanosine-3',5':cyclic monophosphorothioate (100 microM). 4. Inhibition of calcium currents was only partly attenuated by 100 ms depolarizing prepulses to +100 mV immediately preceding the test pulse. However, following occlusion of the sustained depression with okadaic acid (100 nM) the residual kinetic slowing was reversed in a voltage-dependent manner (P < 0.05). 5. Thus pertussis toxin-sensitive G-proteins liberated upon activation of human D2(short) dopamine receptors inhibited high-threshold calcium currents in two distinct ways. The decrease in rate of calcium current activation involved a voltage-dependent pathway, whereas the sustained inhibition of calcium current involved, in part, the voltage-resistant phosphorylation by cyclic AMP-dependent protein kinases and subsequent dephosphorylation by protein phosphatases 1/2A.
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PMID:Phosphorylation- and voltage-dependent inhibition of neuronal calcium currents by activation of human D2(short) dopamine receptors. 758 57

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein phosphatase involvement in the late stages of the secretory pathway for adrenocorticotrophin (ACTH) secretion. The effects of the type 1 and 2 phosphatase inhibitor calyculin A upon calcium-, guanine nucleotide- and phorbol 12-myristate 13-acetate (PMA)-stimulated ACTH secretion from electrically-permeabilized AtT-20 cells were studied. 2. Calyculin A (1 nM-1 microM) inhibited both calcium (10 microM)- and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (100 microM)-evoked ACTH secretion from permeabilized cells in a concentration-dependent manner. These effects were maximal with 100 nM calyculin A. 3. ACTH secretion was stimulated from electrically-permeabilized cells when the cytosolic free calcium ion concentration, controlled by calcium-EGTA buffers, was raised over the concentration range of 100 nM to 10 microM. This calcium-stimulated ACTH secretion was inhibited by co-incubation with calyculin A (100 nM). 4. GTP-gamma-S (10 nM-100 microM) stimulated ACTH secretion from permeabilized cells at concentrations greater than 1 microM GTP-gamma-S. Co-incubation with calyculin A (100 nM) inhibited this stimulation of ACTH secretion observed at these concentrations of GTP-gamma-S. 5. PMA (100 nM) significantly stimulated ACTH secretion from permeabilized cells in the absence of either calcium and guanine nucleotides and this action was enhanced by calyculin A (100 nM). Furthermore, an inhibition of GTP-gamma-S (100 microM)-stimulated ACTH secretion observed in the presence of calyculin A (100 nM) was not observed in the presence of PMA (100 nM). 6. The results of the present study indicate that dephosphorylation by phosphatases plays an important role in stimulus-secretion coupling in AtT-20 cells and is involved in mediating the effects of GE upon the secretory apparatus in these cells. Furthermore, the point of regulation of the secretory response by PKC which underlies the ability of PKC to amplify the calcium/GE system may lie distal to both GE and these phosphatases.
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PMID:The effects of calyculin A upon calcium-, guanine nucleotides- and phorbol 12-myristate 13-acetate-stimulated ACTH secretion from AtT-20 cells. 759 24


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