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 meiotic maturation of Xenopus laevis oocytes is induced in vitro by progesterone which interacts at the cell surface level. A cell-free membrane preparation (P-10,000) incorporated 32P from [gamma-32P]ATP, mostly into two proteins, Mr approximately 56,000 and approximately 48,000 (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Progesterone, added in vitro, specifically inhibited the phosphorylation of the Mr approximately 48,000 protein (named p48). Half-maximal inhibition of p48 phosphorylation occurred with progesterone approximately 8 microM, in good correlation with hormone concentration inducing oocyte maturation. The effect was not due to stimulation of protein phosphatase activity. The potent maturation inducers testosterone and deoxycorticosterone also inhibited p48 phosphorylation, whereas biologically inactive steroids or cholesterol did not. p48 phosphorylation was not affected by cAMP, cGMP, polyamines, calmodulin, and phospholipids + diolein. EGTA had a stimulatory effect which was reversed by added Ca2+. The inhibitory effects of progesterone and Ca2+ were additive, suggesting two distinct sites of action. Phospho-p48 was not detected in yolk platelets, microsomes, and cytosol of oocytes. Contrary to p48 itself, the p48 kinase activity was loosely associated with P-10,000. Progesterone inhibited p48 phosphorylation produced by either cytosol or exogenous pure catalytic subunit of cAMP-dependent protein kinase. Conversely, phosphorylation of casein and histones by protein kinase activity present in P-10,000 was not modified by progesterone. It is then suggested that progesterone regulates p48 phosphorylation by affecting the protein substrate in the membrane, rather than by inhibiting the protein kinase enzyme itself. The data demonstrate a direct effect (not mediated by change of protein synthesis) of steroids on p48 phosphorylation in the plasma membrane, and they suggest that this protein could be implicated in the initial action of progesterone on oocyte maturation.
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PMID:Progesterone-inhibited phosphorylation of an unique Mr 48,000 protein in the plasma membrane of Xenopus laevis oocytes. 298 68

Calmodulin tryptic fragments 78-148, 107-148, and 1-77 coupled to Sepharose 4B were used to test the ability of different calmodulin-regulated enzymes to recognize different domains of calmodulin. Fragment 107-148, which contains a single Ca2+-binding domain, does not interact with any of the calmodulin binding proteins. Fragments 1-77 and 78-148, each of which contains two Ca2+-binding domains, have preserved their ability to interact with several calmodulin-dependent enzymes. Most of the calmodulin-regulated enzymes in brain extracts, such as cAMP phosphodiesterase, cAMP-dependent protein kinase, and the calmodulin-stimulated protein phosphatase (calcineurin) interact with fragment 78-148 in a Ca2+-dependent fashion. An ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive, calmodulin-independent, p-nitrophenyl phosphatase does not bind to the affinity column and is resolved from calcineurin at this step. Although calmodulin-stimulated protein kinase(s) can interact with fragment 78-148, their interaction is prevented by increased ionic strength even in the presence of Ca2+. Fragment 1-77 exhibits a higher degree of selectivity than fragment 78-148. Only cAMP-dependent protein kinase and cAMP phosphodiesterase bind to fragment 1-77. These results confirm the multiple modes of interaction of calmodulin with its target proteins and provide the basis for a selective purification of calmodulin-regulated enzymes by affinity chromatography on specific calmodulin fragments coupled to Sepharose.
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PMID:Selective affinity chromatography with calmodulin fragments coupled to sepharose. 298 37

Occupancy of one of the two phenothiazine-binding sites on calmodulin does not significantly decrease the affinity of calmodulin for its target proteins; however, it does affect the ability of calmodulin to activate some enzymes. Previously we demonstrated that a covalent adduct of calmodulin with one molecule of phenothiazine (CAPP1-calmodulin) is an antagonist for the calmodulin-dependent enzymes, cAMP phosphodiesterase and myosin kinase, and a partial agonist for calcineurin. We now show that CAPP1-calmodulin is a full agonist for glycogen synthase kinase and phosphorylase kinase. Unlike phenothiazines, CAPP1-calmodulin is specific for calmodulin-regulated proteins; it has no effect on protein kinase C. With the exception of phosphorylase kinase, occupancy of two phenothiazine-binding sites completely eliminates the ability of calmodulin to activate these proteins. Thus, the study of the interaction of CAPP1-calmodulin with calmodulin target proteins demonstrates that calmodulin interacts differently with different proteins. This is confirmed by studies of the effect of calmodulin fragments, 1-77 and 78-148, on calmodulin-regulated enzymes.
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PMID:Selective effects of CAPP1-calmodulin on its target proteins. 298 45

The effects of hypothyroidism on glycogen metabolism in rat skeletal muscle were studied using the perfused rat hindlimb preparation. Three weeks after propylthiouracil treatment, serum thyroxine was undetectable and muscle glycogen and Glc-6-P were decreased. Basal and epinephrine-stimulated phosphorylase a and phosphorylase b kinase activities were also significantly reduced, as were epinephrine-stimulated cAMP accumulation and cAMP-dependent protein kinase activity. Conversely, basal and epinephrine-stimulated glycogen synthase I activities were significantly higher while the Ka of the enzyme for Glc-6-P was lower in hypothyroid animals. Propylthiouracil-treated rats also had increased phosphoprotein phosphatase activities towards phosphorylase and glycogen synthase and decreased activity of phosphatase inhibitor 1. beta-Adrenergic receptor binding and basal and epinephrine-stimulated adenylate cyclase activities were reduced in muscle particulate fractions from hypothyroid rats. Administration of triiodothyronine to rats for 3 days after 3 weeks of propylthiouracil treatment restored the altered metabolic parameters to normal. It is proposed that the decreased beta-adrenergic responsiveness of the enzymes of glycogen metabolism in hypothyroid rat skeletal muscle is due to increased activity of phosphoprotein phosphatases and to reduced beta-adrenergic receptors and adenylate cyclase activity.
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PMID:Effects of altered thyroid status on beta-adrenergic actions on skeletal muscle glycogen metabolism. 299 Dec 89

A protein kinase inhibitor was found in rat liver cells as a component of HMG proteins. It is located in cytosol as well as in nuclei. It inhibits all tested cAMP independent protein kinases and has no effect on cAMP dependent protein kinases. This inhibitor is a 25 000 Da protein. It has no ATPase, phosphoprotein phosphatase or proteinase activity and is heat unstable.
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PMID:A 25 000 dalton inhibitor of cAMP independent protein kinases present in rat liver HMG protein preparations. 299 59

The activity of a purified high molecular weight phosphoprotein phosphatase was inhibited by purified type II cAMP-dependent protein kinase. This effect required cAMP and was obtained in the absence of ATP. The isolated type II regulatory subunits (R-subunits) from several species also inhibited the phosphatase activity in both crude extracts and purified preparations. Half maximal inhibition was observed at 0.06-0.25 microM, well within the physiological range of R-subunit concentrations. The inhibitory potency of R-subunit was greater using the thiophosphorylated form. Limited trypsinization of the R-subunit abolished the inhibitory activity. The C-subunit released the bound cAMP when combined with R-subunit, but the phosphatase did not, implying that the inhibited species is a R.cAMP-phosphatase complex. The results suggest that the R-subunit might have at least one physiological role in addition to inhibition of the C-subunit, i.e., inhibition of phosphatase. The latter would occur only when cAMP is elevated.
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PMID:Regulatory subunit of cAMP-dependent protein kinase inhibits phosphoprotein phosphatase. 299 75

The results of the present study permit the explanation of one of the mechanisms of the interconnection between the regulatory systems of cAMP and 2-5A. cAMP-dependent regulation of 2'-PDE was found to involve phosphorylation of the specific protein inhibitor. Originally, a similar way of regulation of the enzyme activity was discovered for protein phosphatase I. This enzyme has a specific protein inhibitor type 1, which is phosphorylated by cAMP-dependent protein kinase and is activated by phosphorylation (18). It is interesting that the molecular weights of 2'-PDE protein inhibitor and of the inhibitor type 1 of protein phosphatase I are essentially the same. There is also a certain similarity between the above described mechanism and phosphorylation of the regulatory subunit of cAMP-dependent protein kinase type 2. The regulatory subunit can also act as a protein inhibitor of the enzyme and change its properties as a result of phosphorylation (19). The results obtained permit as well a more detailed explanation for cAMP-dependent inhibition of cell proliferation. Evidently, cAMP elevation causes activation of cAMP-dependent phosphorylation which, in turn, leads to the induction of 2-5A synthetase and inhibition of 2'-PDE. As a result of variations in the activities of these enzymes, the level of 2-5A rises. The latter brings about the changes characteristic of the resting state. They involve activation of RNase L and the succeeding acceleration of RNA hydrolysis, inhibition of protein synthesis and cell proliferation. The resting state is characterized by a rapid turnover of macromolecules due to their intensive degradation (20). The above described scheme suggested that the rapid turnover of RNA during inhibition of cell proliferation can be partially accounted for by activation of 2-5A-dependent RNase L. Thus, it can be thought that at least one of the mechanisms of the antiproliferative effect of cAMP-dependent phosphorylation of proteins involves cAMP-dependent elevation of intracellular 2-5A. Evidently, a number of properties of the resting cells are determined by the elevated content of 2-5A. Finally, it should be noted that the interconnection between the systems of cAMP and 2-5A is a multiple process. We have earlier demonstrated (12) that 2-5A activates cAMP phosphodiesterase in NIH 3T3 cell homogenates. These data suggest that the mutual regulation of cAMP and 2-5A levels involves the negative feedback mechanism (Fig. 8).
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PMID:Regulation of 2-5 A phosphodiesterase activity by cAMP-dependent phosphorylation: mechanism and biological role. 300 Jan 46

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and characterization of calcineurin from bovine brain. 300 May 60

The dissociated regulatory subunit (RII) of autophosphorylated cAMP-dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase-1 and -2A (phosphatase-1c and -2Ac) and by a high-Mr polycation-dependent form of phosphatase-2A (2Ao) with Km values of 5, 0.3 and 1 microM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase-1c, whereas polycations (histone Hl or polybrene) markedly stimulated phosphatase-2Ac and -2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half-maximum inhibitory concentrations of 0.1-0.36 microM.
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PMID:Regulatory subunit of type II cAMP-dependent protein kinase as substrate and inhibitor of protein phosphatase-1 and -2A. 300 38

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.
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PMID:Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase. 300 26


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