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

---

Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The effects of the protein phosphatase inhibitors okadaic acid and microcystin LR on the regulation by insulin of pyruvate dehydrogenase and acetyl-CoA carboxylase have been studied in rat epididymal fat-pads and isolated cells. These inhibitors both completely blocked the phosphatase activity (against phosphorylase a) present in extracts of epididymal fat-pads, with half-maximal effects in the nanomolar range. 2. Okadaic acid treatment of pads and cells lowered the activity of acetyl-CoA carboxylase assayed in tissue extracts, both before and after treatment of the extracts with the activator, citrate. Further, okadaic acid treatment abolished the 2-3-fold difference in activity observed between extracts from control and insulin-treated tissues, assayed without prior treatment with citrate. 3. Incubation of pads with [32P]Pi, sufficient to label the intracellular pool of ATP, demonstrated that okadaic acid increased the overall phosphorylation of acetyl-CoA carboxylase on a number of distinct sites, as judged by two-dimensional mapping of tryptic peptides. These included the 'I-peptide' [Brownsey & Denton (1982) Biochem. J. 202, 77-86], the phosphorylation of which may be associated with the stimulation of the activity of the enzyme by insulin, as well as inhibitory phosphorylation sites. 4. Incubation with 1 microM-okadaic acid had no effect on the basal level of active pyruvate dehydrogenase apparent after tissue extraction, but abolished the 2-3-fold increase in this parameter which was elicited by insulin in the absence of okadaic acid. However, okadaic acid treatment did not affect the persistent increase in active pyruvate dehydrogenase levels which was apparent in mitochondria subsequently isolated from insulin-treated pads and re-incubated with an oxidizable substrate. It is concluded that the effects of okadaic acid are exerted through changes in metabolite concentrations rather than some direct action on the signalling pathway whereby insulin stimulates pyruvate dehydrogenase. 5. Microcystin LR did not mimic the effects of okadaic acid on intact cells and pads described above.
...
PMID:Effects of protein phosphatase inhibitors on the regulation of insulin-sensitive enzymes within rat epididymal fat-pads and cells. 167 87

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.
...
PMID:Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion. 167 49

To determine the role of protein phosphorylation in neutrophil activation, electropermeabilized cells were treated with vanadate, a phosphatase inhibitor. Micromolar concentrations of vanadate elicited a NADPH-dependent burst of oxygen utilization in permeabilized, but not in intact cells, indicating an intracellular site of action. Stimulation of oxygen consumption by vanadate was reversible, concentration dependent and required the presence of ATP and Mg2+. Generation of a respiratory burst by vanadate was associated with accumulation of phosphorylated proteins. Such accumulation was due, at least in part, to inhibition of phosphoprotein phosphatase activity, as indicated by pulse-chase experiments. No evidence for stimulation of protein kinases by vanadate was found. Phosphoamino acid analysis revealed that a large fraction of the vanadate-induced phosphorylation occurred on tyrosine residues. The pronounced accumulation of tyrosine-phosphorylated proteins was confirmed by immunoblotting with anti-phosphotyrosine antibodies. The data suggest that neutrophils possess one or more constitutively active tyrosine kinases and that phosphoprotein accumulation is normally prevented by vigorous concomitant phosphatase activity. Inhibition of the latter by vanadate leads to phosphoprotein accumulation and is accompanied by stimulation of oxygen consumption.
...
PMID:Vanadate stimulates oxygen consumption and tyrosine phosphorylation in electropermeabilized human neutrophils. 168 31

A combination of planar bilayer and patch-clamp techniques was used to determine whether apical membrane Cl- channels of shark (Squalus acanthias) rectal gland (SRG) were regulated by a phosphorylating and dephosphorylating cycle. In channel reconstitution studies, apical membrane vesicles of SRG were purified, incubated in ATP-Mg2+ and the presence or absence (control) of catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAMP-PK) and incorporated into planar lipid bilayers. In the presence of cAMP-PK, two distinct Cl- channels were found when imposing either 450/50 or 300/50 mM KCl (cis/trans) gradients. The most frequently observed channels (G beta 1) were open greater than 80% at all potentials between -60 and +20 mV (trans ground) and were inactivated by alkaline phosphatase added to the cis chamber. The single-channel conductance of G beta 1 was 42 pS between -60 and +20 mV with a 300/50 mM KCl gradient. The second channel (G beta 2) was always observed in pairs of 62-pS subchannels and was not affected by alkaline phosphatase, but the open probability increased with depolarizing potentials. G beta 2 was observed once, but G beta 1 was never observed in the absence of cAMP-PK. In parallel patch-clamp studies of the apical membrane of cultured SRG, a 50-pS channel similar to G beta 1 was noted after incubating cells with either forskolin, an activator of adenylate cyclase, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A. It is concluded that G beta 1 of SRG can be studied in both patch-clamp and bilayer preparations and that G beta 1 is regulated by reversible phosphorylation by cAMP-PK and dephosphorylation by a protein phosphatase.
...
PMID:Regulation of epithelial chloride channels by protein phosphatase. 171 76

In order to identify the endogenous phosphoprotein substrates for human prostatic acid phosphatase (PAP), cellular proteins of human normal, benign, and malignant prostatic tissues as well as carcinoma cell lines were phosphorylated by the cellular kinases in the presence of (gamma-32P)-ATP and then were subjected to dephosphorylation reaction by PAP. Of several endogenous phosphoproteins, PAP preferentially dephosphorylated a cytosolic protein of Mr 83 kDa. The dephosphorylation of the 83 kDa phosphoprotein (designated pp83) by PAP was uniformly observed in all cells/tissues of prostate origin, and was completely inhibited by L(+)-tartrate, the classic inhibitor of PAP. Phosphoamino acid analysis revealed that pp83 was a tyrosine-poor phosphoprotein and was mostly dephosphorylated by PAP at serine/threonine residues rather than tyrosine residues. Further comparison of dephosphorylation rate with that of an endogenous phosphotyrosine-containing phosphoprotein (pp53) revealed that PAP possessed both phosphoserine/threonine protein phosphatase and phosphotyrosine protein phosphatase activity. These results demonstrate that pp83 apparently is an endogenous substrate of PAP in human prostate, and that, instead of a phosphotyrosine protein specific phosphatase, PAP is a universal protein phosphatase hydrolyzing equally well the phosphotyrosine, serine, and threonine residues.
...
PMID:Endogenous protein substrates for prostatic acid phosphatase in human prostate. 171 11

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.
...
PMID:A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts. 171 69

Adenylylcyclase activity in the flagella of gametes of Chlamydomonas reinhardtii was inhibited by prior incubation at or below 30 degrees C in the presence of ATP. This decrease did not occur in the absence of ATP, in the presence of the ATP analog 5'-adenylylimidodiphosphate (App(NH)p), or in the presence of ATP plus the protein kinase inhibitor staurosporine (2 microM). If ATP treatment was performed in the absence of an ATP-regenerating system, activity initially declined and subsequently recovered. Incubation of flagella at 45 degrees C in the absence of ATP or incubation at lower temperatures in the presence of either App(NH)p or staurosporine both increased adenylylcyclase activity (over 10-fold) and blocked subsequent ATP-dependent loss of activity at 30 degrees C. This heat-induced activation was prevented by the presence of ATP plus an ATP-regenerating system. Incubation of flagella with [gamma-32P]ATP followed by gel electrophoresis in sodium dodecyl sulfate indicated the presence of endogenous protein kinase and protein phosphatase activities. These data suggest that the flagellar adenylylcyclase in Chlamydomonas gametes is inhibited by phosphorylation and stimulated by dephosphorylation. This mechanism for regulating adenylylcyclase may underlie the rapid increase in cyclic AMP that is induced by flagellar adhesion during fertilization in Chlamydomonas.
...
PMID:ATP-dependent regulation of flagellar adenylylcyclase in gametes of Chlamydomonas reinhardtii. 174 89

Several rat liver HMG-CoA-reductase (HMG-CoA-Rd) phosphatase activities have been shown to be associated with the endoplasmic reticulum. These activities were not due to glycogen contamination, as judged not only from different patterns of solubilization of the microsomal membranes and the glycogen pellet but also by differential centrifugation behavior under standard conditions and in a sucrose gradient. We present evidence that at least three forms of protein phosphatase are associated with microsomal membranes: a polycation-stimulated type 2A phosphatase, a type 2C phosphatase, and a non-2A, non-2B, non-2C phosphatase. This last HMG-CoA-Rd phosphatase activity corresponding to an 85 kDa protein was partially purified by several chromatographic procedures. The IC50 value for the inhibition of the HMG-CoA-Rd phosphatase by I-2 was 10-fold higher than for the inhibition of the purified type 1 catalytic subunit from rabbit skeletal muscle. The microsomal HMG-CoA-Rd phosphatase activity was slightly affected by the protein inhibitor that inhibits type 2A activity when HMG-CoA reductase is the substrate. The HMG-CoA-Rd phosphatase activity is spontaneously active and it is not reactivated in the presence of Mg2+ or polycations. The holoenzyme does not contain the inhibitor-2 and it is not reactivated by incubation with ATP and glycogen synthase kinase-3. Proteolytic treatment of the enzyme yielded a polypeptide fragment of low Mr (37 kDa) with reduced activity. A model of holoenzymatic HMG-CoA-Rd phosphatase and its relation to the microsomal membranes is presented.
...
PMID:Isolation and partial characterization of a protein with HMG-CoA reductase phosphatase activity associated with rat liver microsomal membranes. 175 9

The direct effect of okadaic acid (OA) on the ATP-dependent interaction between actin and myosin of smooth muscle was examined not only by the conventional measurement of ATPase activity but also by application of in vitro motility assay developed recently. The motility was effectively enhanced by microM levels of OA. Measurements of the activities of myosin confirmed that the myosin mediated this effect. The result of this study, which was carried out in the absence of protein phosphatase, are not compatible with the recent reports that the stimulatory effect of OA on smooth muscle contraction is attributable to its inhibitory effect on the activity of the protein phosphatase.
...
PMID:Okadaic acid stimulates the ATP-dependent interaction between actin and myosin of smooth muscle via a direct effect on myosin. 182 55

1. In the adipocyte, phosphorylation/dephosphorylation of regulatory proteins is a common mechanism of metabolic regulation. We have observed a very prominent phosphoprotein doublet of 61 kDa and 63 kDa in rat adipocytes that is markedly responsive to hormones. The 63 kDa band was the predominant phosphoprotein in the cell in response to 0.1 microM-isoprenaline, whereas the 61 kDa band was nearly absent. Insulin alone did not alter 32P incorporation into the doublet, but partially counteracted the effects of isoprenaline, decreasing label in the 63 kDa band by as much as 50% and resulting in the reappearance of the 61 kDa band. 2. Subcellular fractionation demonstrated that both phosphoprotein bands were fat-associated. Neither insulin nor isoprenaline altered this localization. Peptide maps (one-dimensional) of the 61/63 kDa bands demonstrated close sequence similarity. Amino acid analysis revealed the presence of phosphoserine and phosphothreonine. The latter was more prominent in the 61 kDa band. Isoprenaline caused an absolute increase in both phosphoamino acids. 3. Permeabilization of 32P-labelled isoprenaline-treated cells with digitonin initiated rapid dephosphorylation of the 63 kDa band, with reappearance of the 61 kDa band. Insulin increased the rate of dephosphorylation by 2-3-fold when present with isoprenaline before permeabilization. 4. In permeabilized adipocytes, cyclic AMP (1 microM-1 mM) increased phosphorylation of the 61/63 kDa doublet by 4-10-fold in the presence of [gamma-32P]ATP, but insulin had no effect. 5. We conclude that this prominent phosphoprotein, migrating as a 61/63 kDa doublet, is coupled to the cyclic AMP-dependent protein kinase and is associated with an insulin-stimulated phosphoprotein phosphatase activity. This fat-associated phosphoprotein, which is under counter-regulatory hormonal control, may play a role in hormone-dependent lipid metabolism.
...
PMID:Counter-regulation by insulin and isoprenaline of a prominent fat-associated phosphoprotein doublet in rat adipocytes. 184 60


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---