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)

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.
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PMID:Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle. 352 89

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

Polyomavirus-infected cells express three proteins in the early phase of the lytic cycle, the so-called tumor antigens. Two of them, large- and middle-T antigens, are also required for virus-mediated transformation of primary cells, while middle-T alone is sufficient to transform established cells in culture. Cell transformation by middle-T is strictly dependent on the ability of this protein to associate with cellular enzymes like members of the Src family of tyrosine kinases, a phosphatidylinositol 3-kinase, phosphatase 2A and SHC, an adapter protein linking GDP/GTP exchange factors to tyrosine kinase receptors. A carboxy-terminal stretch of 22 hydrophobic amino acids is required for targeting middle-T and associated proteins to cellular membranes. Here we show in an in vitro system that middle-T fusion proteins carrying an amino-terminal hemagglutinin leader sequence are capable to bind to and enter the lumen of dog pancreas microsomes supporting the concept that the carboxy-terminus of middle-T is an authentic membrane-targeting domain. Furthermore, wild-type middle-T, but not a truncated protein lacking the putative membrane anchor, specifically associates with artificial lipid bilayers.
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PMID:Membrane association of polyomavirus middle-T antigen in an in vitro system. 776 90

The specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study differentiation-dependent changes in the regulation of the low-molecular-mass GTP-binding protein Rho. Differentiation of F9 teratocarcinoma cells to neuronal-like cells by treatment with retinoic acid and dibutyryl-adenosine 3',5'-monophosphate [(Bt)2cAMP] increased the C3-catalyzed ADP-ribosylation of RhoA proteins in cytosolic and membrane fractions by about threefold and sixfold, respectively. Phenotypical differentiation of F9 cells was not required for increase in ADP-ribosylation. Increase in ADP-ribosylation after (Bt)2cAMP and retinoic acid treatments was blocked by cycloheximide, indicating the requirement of protein biosynthesis. As deduced from specific rho mRNA amounts and from Western analysis with a monoclonal RhoA antibody, the stimulation in the [32P]ADP-ribosylation of Rho was not caused by an increased de-novo synthesis of Rho proteins. GDP increased the ADP-ribosylation of membrane-associated Rho from non-differentiated, but not from differentiated F9 cells. GTP[S] decreased ADP-ribosylation of membranous Rho from differentiated and much less from non-differentiated F9 cells. Differentiation-dependent increase in ADP-ribosylation of cytosolic Rho was reversed by protein phosphatase type-1. Treatment with SDS (0.01%) which releases Rho from complexation with guanine nucleotide dissociation inhibitor, increased ADP-ribosylation both in differentiated and non-differentiated cells, indicating no differentiation-specific change of such complexes. In total, our data indicate that the induction of the differentiation process in F9 cells is accompanied by changes in the regulation of cytosolic and membrane-associated Rho proteins.
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PMID:Differentiation-induced increase in Clostridium botulinum C3 exoenzyme-catalyzed ADP-ribosylation of the small GTP-binding protein Rho. 805 68

Thrombin dramatically activated p72syk in a time- and dose- dependent fashion in extracts of resting porcine platelets in the presence of EDTA. Separation analysis using Sephacryl S-300 column chromatography has demonstrated that p72syk may exist as large (complex) and small (monomer) forms in resting platelets, and activation of p72syk was only observed in the fraction of large form. Pretreatment with ATP scavenger, GDP beta S and protein phosphatase inhibitors had no effect on this activation. Furthermore, washed immuno-precipitates of large form p72syk were also activated by thrombin or fibrinogen. These results suggest that p72syk may associate with thrombin receptor or other agonist receptors and there may be a novel activation mechanism of non-receptor type protein-tyrosine kinase, which does not require the modification by other protein kinases, protein phosphatases and GTP binding proteins.
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PMID:Activation of p72syk by thrombin in a cell-free system. 816 76

The voltage-dependent Ca2+ channels (VDC) in smooth muscle cell membranes are the major pathway by which Ca2+ enters the cell during contraction. It has been reported that VDC can be modulated by reversible channel protein phosphorylation and dephosphorylation reactions. In intestinal smooth muscle cell, muscarinic agents have been reported to increase the break-down of phosphatidylinositol 4,5-bisphosphate, which indicates that inositol-1,4,5-trisphosphate and diacylglycerol (DG) can be generated. DG activates protein kinase C. Carbachol (CCh), phorbol 12,13-dibutyrate and phosphatase inhibitors, okadaic acid and calyculin A increased the inward currents passing through the L-type VDCs. These effects were inhibited by protein kinase inhibitors, H-7 and staurosporine. The CCh effect was also inhibited by GDP beta S. Therefore, it seems possible that DG, a product of phosphatidylinositol break-down might mediate muscarinic effects on L-type VDC, the last via stimulation of protein kinase C, and that L-type VDC activity might be modulated by protein kinase C-mediated phosphorylation and protein phosphatase type-1-mediated dephosphorylation of the channel or related protein(s) in guinea pig taenia coli smooth muscle cells.
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PMID:[Modulation of the voltage-dependent Ca2+ channels of smooth muscle by phosphorylation]. 838 50

Plasma membrane-enriched fractions were prepared from human embryonic retinal cells transformed with either adenovirus E1A and oncogenic ras DNA, or E1A and E1B DNA. Ras comprised 5-10% of the membrane protein from the E1A/ras transformed cells, whereas the membranes from E1A/E1B transformed cells did not overexpress Ras. The membranes from E1A/ras cells contained MAP kinase kinase kinase (MAPKKK) activity, even after washing in 0.5 M NaCl, whereas the membranes from E1A/E1B cells did not. Neither membrane fraction contained MAP kinase kinase or MAP kinase activity after washing with 0.5M NaCl. Immunoblotting experiments revealed about 10-fold more c-Raf in the membranes from E1A/ras cells than from E1A/E1B cells, and 50-60% of the MAPKKK activity in Triton X100-solubilised membranes from E1A/ras cells was immunoprecipitated with anti-Raf antibodies. A striking enrichment of c-Raf in the plasma membranes of E1A/ras cells was also demonstrated by immunocytochemistry, where it was co-localized with Ras. The MAPKKK activity in E1A/ras membranes was unaffected by incubation with protein phosphatases or by inclusion of protein phosphatase inhibitors during isolation, nor was it activated by GTP-Ras or inhibited by GDP-Ras. The results support the view that Ras and c-Raf interact with one another, but that neither c-Raf phosphorylation nor its interaction with GTP-Ras are alone sufficient for activation. The identification of MAPKKK activity in the membranes of ras-transformed cells may prove useful in elucidating the mechanism by which Raf is activated by Ras.
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PMID:Specific association of activated MAP kinase kinase kinase (Raf) with the plasma membranes of ras-transformed retinal cells. 841 21

1. We used the patch-clamp technique, in conjunction with membrane capacitance measurement, fluorescence measurement of intracellular calcium concentration ([Ca(2+)](i)), and flash photolysis of caged Ca(2+) to study exo- and endocytosis in identified rat corticotrophs. 2. Exocytosis stimulated by depolarization pulses was typically followed by a 'slow' endocytosis that retrieved the membrane with a time constant of approximately 6 s. The efficiency (the endocytosis/exocytosis amplitude ratio) of 'slow' endocytosis was approximately 1.2 at [Ca(2+)](i) < 3 microM and increased to approximately 1.6 at [Ca(2+)](i) > 3 microM. 3. Whole-cell dialysis through a patch pipette did not affect the kinetics and the efficiency of 'slow' endocytosis, but the amplitude of exocytosis was reduced. 4. 'Slow' endocytosis did not require sustained [Ca(2+)](i) elevation and its kinetics was only weakly [Ca(2+)](i) dependent. Our results suggest that 'slow' endocytosis involves a Ca(2+) sensor with a high Ca(2+) affinity (approximately 500 nM). 5. At high [Ca(2+)](i) (> 10 microM), the 'slow' endocytosis was frequently preceded by a 'fast' endocytosis that comprised multiple steps of rapid decrease in membrane capacitance. 6. Neither calmodulin nor calcineurin appeared to be the Ca(2+) sensor for endocytosis because the two forms of endocytosis were not affected by the calmodulin inhibitor calmidazolium (500 microM) or the calcineurin inhibitors cyclosporin A (1 microM) and calcineurin autoinhibitory peptide (1 mg ml(-1)). Ba(2+), a poor activator of calmodulin, could support both forms of endocytosis but slowed the kinetics of 'slow' endocytosis approximately 2-fold. 7. Non-hydrolysable analogues of GTP (GDP-beta-S) and ATP (ATP-gamma-S) also failed to inhibit either form of endocytosis, indicating that neither GTP nor ATP was essential for endocytosis. 8. We suggest that the high Ca(2+) affinity of 'slow' endocytosis may be important for maintaining continuous cycles of exocytosis-endocytosis during sustained adrenocorticotropin secretion in corticotrophs.
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PMID:Endocytosis in identified rat corticotrophs. 1138

The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein.
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PMID:Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors. 1222 6

The small GTPase Ran functions in several critical processes in eukaryotic cells including nuclear transport, nuclear envelope formation, and spindle formation. A RanGDP-binding protein, NTF2, facilitates translocation of RanGDP through the nuclear pore complex and also acts to stabilize RanGDP against nucleotide exchange. Here, we identify a novel activity that stimulates release of GDP from Ran in the presence of NTF2. Hydrolyzable ATP enhances the GDP dissociation activity, and this enhancement is inhibited by nonhydrolyzable ATP analogues. In contrast, neither hydrolyzable ATP nor nonhydrolyzable ATP analogues affect GDP dissociation from Ran catalyzed by recombinant RCC1 or inhibition of GDP dissociation from Ran by recombinant NTF2. The ATP-dependent RanGDP dissociation activity therefore has the properties of a RanGDP dissociation inhibitor (GDI) displacement factor (RanGDF) where the GDI is NTF2. A protein phosphatase inhibitor mixture stimulates the RanGDF activity, suggesting the activity is regulated by phosphorylation. We propose that the ATP-dependent NTF2 releasing factor may have a role in the RanGDP/GTP cycle.
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PMID:An ATP-dependent activity that releases RanGDP from NTF2. 1515 37


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