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)

The expressed catalytic activity of liver microsomal HMG CoA reductase, the limiting enzyme in cholesterol synthesis, is reversibly diminished by phosphorylation in vitro. In intact hepatocytes the expressed activity of HMG CoA reductase is enhanced by incubation of cells with insulin, and diminished by treatment with glucagon or with mevalonate. In the latter situations the level of total reductase activity falls following initial inactivation (phosphorylation) of the enzyme. This observation suggested that the phosphorylated form of HMG CoA reductase is more sensitive to proteolysis. HMG CoA reductase is a 97,000 dalton (97 K) integral protein of the endoplasmic reticulum with a cytosolic domain that includes the catalytic site and serine residues that may be reversibly phosphorylated. In vitro the Ca2+-activated proteolytic enzyme, calpain, generates two catalytically-active fragments: a membrane bound 62 K and a soluble 53 K form of the enzyme which are quantified by specific immunoblot procedures. Cleavage of the native 97 K HMG CoA reductase is enhanced by pretreatment (inactivation) of microsomes with ATP (Mg2+) and liver reductase kinase compared to microsomes pretreated with protein phosphatase. This is reflected in a loss of 97 K reductase and an increase in the soluble 53 K form of the enzyme. Degradation of HMG CoA reductase in hepatocytes is partially blocked by lysosomotropic agents and insulin. A steady state model for the turnover of proteins subject to reversible phosphorylation has been developed which recognizes fractional degradative rate constants for the phosphorylated and dephosphorylated species.
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PMID:Phosphorylation state of HMG CoA reductase affects its catalytic activity and degradation. 302 50

The N-terminal part sequences of pituitary growth hormone, N alpha-acetyl-hGH 7-13 and hGH 6-13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.
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PMID:Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity. 303 64

A new procedure has been developed for identifying phosphoserine residues in proteins, and is used to analyse the in vivo phosphorylation state of inhibitor-2. The method employs reverse-phase liquid chromatography to resolve phosphorylated and dephosphorylated forms of peptides and fast-atom bombardment mass spectrometry (FABMS) to identify phosphorylated derivatives. The positions of phosphorylation sites within peptides are located by gas-phase sequencer analysis after conversion of phosphoserine residues to S-ethylcysteine. The phosphorylation sites on inhibitor-2 were identified as serines-86, -120 and -121, the three residues phosphorylated in vitro by casein kinase-II. Serine-86 was phosphorylated to 0.7 mol/mol and serines-120 and -121 each to 0.3 mol/mol. These values were not altered significantly by intravenous injection of adrenalin or insulin. No phosphate was present in the region comprising residues 1-49, even after injection of adrenalin, demonstrating that inhibitor-2 is not a substrate for cyclic AMP-dependent protein kinase in vivo. The absence of phosphotyrosine also indicated that inhibitor-2 is not a physiological substrate for the insulin receptor. Surprisingly, no phosphate was present at threonine-72, the residue phosphorylated in vitro by glycogen synthase kinase-3, after injection of either propranolol, adrenalin or insulin. The implications of this finding for the in vivo activation of protein phosphatase 1I (the 1:1 complex between inhibitor-2 and the catalytic subunit of protein phosphatase-1) are discussed. FABMS analysis of inhibitor-2 confirmed the accuracy of the primary structure reported previously, and showed that the only post-translational modifications were an N-acetyl moiety and the three phosphoserine residues. FABMS also demonstrated the presence of an additional serine residue at the C-terminus, and showed that 50% of isolated inhibitor-2 molecules lack the C-terminal Ser-Ser dipeptide.
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PMID:Analysis of the in vivo phosphorylation state of protein phosphatase inhibitor-2 from rabbit skeletal muscle by fast-atom bombardment mass spectrometry. 303 52

Branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase in isolated rat pancreatic islets were shown to be regulated by a phosphorylation/dephosphorylation mechanism. Broad-specificity phosphoprotein phosphatase treatment stimulated and ATP addition inhibited their activities. The kinases responsible for inactivating these complexes were shown to be sensitive to inhibition by known inhibitors, alpha-chloroisocaproate and dichloroacetate. Total activity (nmol/min/islet / 37 degrees C) of branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase was 0.86 and 5.09, with a % active form (activity before phosphatase treatment divided by activity after phosphatase treatment X 100) of 36% and 94%, respectively. Incubation of intact isolated islets with alpha-chloroisocaproate affected neither insulin release nor flux through branched-chain alpha-ketoacid dehydrogenase.
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PMID:Branched chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase activity in isolated rat pancreatic islets. 304 32

We recently proposed a hypothesis for the molecular mechanism of the osteogenic action of fluoride in which it stimulates osteoblast proliferation via the inhibition of an osteoblastic acid phosphatase-like phosphotyrosyl protein phosphatase activity. To test this hypothesis, we investigated whether orthovanadate, a known phosphotyrosyl protein phosphatase inhibitor, would mimic fluoride in the stimulation of bone cell proliferation and bone collagen synthesis in vitro. Orthovanadate inhibited the osteoblastic acid phosphatase activity and stimulated bone cell proliferation at the same low concentrations (i.e. 5-15 microM). At the mitogenic doses, orthovanadate also showed a dose-dependent increase in alkaline phosphatase (a marker of mature osteoblasts) in cultured calvarial cells and stimulated bone collagen synthesis, as measured by the incorporation of [3H]proline and the conversion into [3H] hydroxyproline in organ calvaria cultures. Therefore, orthovanadate stimulated bone formation by increasing the number of mature osteoblasts mediated via stimulation of cell proliferation and differentiation. Orthovanadate was dependent on the presence of a mitogen in cell medium for its mitogenic action in vitro and synergistically potentiated the mitogenic actions on osteoblasts of those growth factors, i.e. insulin, epidermal growth factor, insulin-like growth factor I, and skeletal growth factor, whose mitogenic action involved tyrosyl protein phosphorylation. However, the interaction between orthovanadate and basic fibroblast growth factor, a growth factor that does not appear to involve tyrosyl protein phosphorylation, on bone cell proliferation was additive. In summary, these data are consistent with the hypothesis that inhibition of the osteoblastic phosphotyrosyl protein phosphatases can prolong and/or potentiate the mitogenic actions of growth factors, and thereby stimulates cell proliferation.
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PMID:Vanadate stimulates bone cell proliferation and bone collagen synthesis in vitro. 305 61

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

A polypeptide from a tryptic digest of bovine serum albumin potentiates glucose oxidation stimulated by insulin in isolated rat adipocytes. We studied whether this effect is related to a modification of the insulin receptor kinase. In a solubilized rat adipocytes receptor system, the peptide caused dose-dependent inhibition of the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of insulin receptor. The peptide also inhibited stimulation by vanadate of tyrosine autophosphorylation of the beta subunit of the receptor, though it enhanced vanadate-stimulated glucose oxidation. During the phosphorylation reaction, no phosphorylated forms of the peptide could be detected. The peptide had no effect on dephosphorylation of the phosphorylated beta subunit of the insulin receptor. These results strongly suggest that the inhibition of phosphorylation by the peptide is due not to either simple substrate competition or activation of phosphoprotein phosphatase, but to specific inhibition of tyrosine-specific protein kinase.
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PMID:Inhibition of tyrosine autophosphorylation of the solubilized insulin receptor by an insulin-stimulating peptide derived from bovine serum albumin. 355 83

The relationship between cAMP-dependent protein kinase (A-kinase) activity ratios and lipolysis in the presence of insulin was compared to the standard relationship between these two parameters established with a variety of adenylate cyclase modulators (Honnor, R. C., Dhillon, G., and Londos, C. (1985) J. Biol. Chem. 260, 15130-15138). Three phases of insulin action were observed. First, when tested in control cells exhibiting A-kinase activity ratios up to approximately 0.25, insulin inhibition of lipolysis could be accounted for by the decrease in A-kinase activity. Second, in cells exhibiting A-kinase activity ratios greater than 0.3, the decrease in kinase activity by insulin did not account for the decrease in lipolysis. Finally, as the A-kinase activity ratio approached 0.6 the insulin effect on lipolysis was lost. The data suggest that protein phosphatase activation accounts for the cAMP-independent insulin action. Moreover, the insulin effect not accounted for by a decrease in A-kinase activity appears to be elicited only upon elevation of A-kinase activity. The method by which cells were stimulated determined the IC50 for insulin inhibition of: 1) A-kinase activity ratios, 2) lipolysis explained by the decrease in A-kinase activity ratios, and 3) lipolysis not explained by a decrease in A-kinase activity ratios. For all three parameters, cells stimulated by lipolytic hormones were approximately 5 times more sensitive to insulin than cells stimulated by incubation in a ligand-free environment achieved with adenosine deaminase; insulin IC50 values were approximately 120 and 600 pM, respectively. Such data establish a link between insulin actions in modifying cAMP concentrations and in modifying events apparently independent of changes in cAMP. It is proposed that the receptors and regulatory components associated with adipocyte adenylate cyclase are associated also with components of the insulin response system separate from cyclase.
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PMID:cAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators. 390 91

The effects of amiloride and of natural aliphatic polyamines on basal and hormone-stimulated protein phosphorylations in hepatocytes were studied. Cells isolated from adult rats were incubated in suspension with (32P)-orthophosphate, in the absence or presence of the effectors at varying concentrations and for different times; hepatocytes were then exposed to various hormones for 10 min. Phosphoproteins contained in total cell lysates were analyzed by one- and two-dimensional gel electrophoresis and autoradiography. Amiloride and spermine (the most effective amine) decreased the basal level of phosphorylation of proteins of 46, 34 and 22 kDal, and increased that of 18 kDal and 93 kDal proteins. These effects were maximal with external concentrations of 1 mM and 7.5-10 mM amiloride and spermine, respectively. They were detectable after a lag period of about 10 min and reached a plateau after 45 min. Pretreatment of cells with these effectors almost completely prevented stimulation of the phosphorylation of the 46 and 34 kDal proteins by insulin. In contrast, the effects of vasopressin on the same proteins were only partly inhibited, whereas those of glucagon appeared largely unaffected. The major effect observed in intact cells (i.e., decreased phosphorylation) could be reproduced in a cell-free system where no kinase activity persisted. Amiloride or spermine added directly to cell extracts strongly accelerated the dephosphorylation of 46 kDal protein and also of the 61 kDal protein identified as pyruvate kinase. Furthermore, restoration of the activity of this enzyme occurred concomitantly with dephosphorylation of the 61 kDal protein, an observation supporting the notion that amiloride and spermine could activate a phosphoprotein phosphatase.
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PMID:Insulin regulation of protein phosphorylation in hepatocytes. Studies using two effectors: amiloride and natural aliphatic polyamines. 390 16

1. The activity of acetyl-CoA carboxylase (EC 6.4.1.2) in extracts of freeze-clamped liver samples from fed or 24 h-starved virgin, pregnant, lactating and weaned rats was measured (i) immediately after preparation of extracts (;I activity'), (ii) after incubation of extracts with partially purified preparations of either rabbit muscle protein phosphatase 1 [Antoniw, Nimmo, Yeaman & Cohen (1977) Biochem. J.162, 423-433] or rabbit liver phosphatase [Brandt, Capulong & Lee (1975) J. Biol. Chem.250, 8038-8044] (;A activity') and (iii) after incubation with 20mm-potassium citrate before or after incubation with phosphatases (;C activity'). 2. Incubation of liver extracts at 30 degrees C without any additions resulted in activation of acetyl-CoA carboxylase that was shown to be due to dephosphorylation of the enzyme by endogenous protein phosphatase activity. This latter activity was not stimulated by Ca(2+) and/or Mg(2+) but was stimulated by 1 mm-Mn(2+). Incubation of extracts with either of the partially purified phosphatases (0.2-0.5 unit) resulted in faster dephosphorylation and activation. The activity achieved after incubation with either of the exogenously added phosphatases was similar. 3. The A and C activities increased during late pregnancy, were lower than in the virgin rat liver during early lactation and increased by 2-fold in liver of mid-lactating rats. Weaning of mid-lactating rats for 24 h resulted in no change in A and C activities but after 48 h weaning they were significantly lower than those in livers from suckled mothers. 4. The I activity followed a similar pattern of changes as the A and C activities during pregnancy and lactation such that, although the I/A and I/C activity ratios tended to be lower during late pregnancy and early lactation, there were no significant changes in I/A and I/C ratios between lactating and virgin animals. However, these ratios were significantly higher in liver from fed 24 h-weaned animals. 5. Starvation (24 h) resulted in a marked decrease in I activity for all animals studied except early-lactating rats. This was due to a combination of a decrease in the concentration of acetyl-CoA carboxylase in liver of starved animals (A and C activities) and a decrease in the fraction of the enzyme in the active form (lower I/C and I/A ratios). The relative importance of the two forms of regulation in mediating the starvation-induced fall in I activity was about equal in livers of virgin, pregnant and lactating animals. However, the decrease in I/A and I/C ratios was of dominating importance in livers of weaned animals. The A/C activity ratios were the same for livers from all animals studied. 6. The maximal activity of fatty acid synthase was also measured in livers and was highly and positively correlated with the A and C activities of acetyl-CoA carboxylase, suggesting that the concentrations of the two enzymes in the liver were controlled coordinately. 7. It is suggested that the lack of correlation between plasma insulin levels and rates of lipogenesis in the transition from the virgin to the lactating state may be explained by different effects of insulin and prolactin on the concentration of acetyl-CoA carboxylase in the liver and on the fraction of the enzyme in the active form.
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PMID:Changes in the proportion of acetyl-CoA carboxylase in the active form in rat liver. Effect of starvation, lactation and weaning. 612 71


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