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)

Two isoforms of calcineurin beta subunit(beta 1 and beta 2) were identified in rat testis by a monoclonal antibody Va1. Both beta 1 and beta 2 were recovered in calmodulin binding protein fraction and showed calcium shift on SDS-polyacrylamide gel electrophoresis which is the specific character for EF-hand calcium binding protein. beta 2 showed same apparent molecular weight on SDS-PAGE as that of brain calcineurin beta and was found in wide variety of tissues. beta 1 was shown to have six amino acid polypepeptide sequence and it showed higher molecular weight than brain beta and was specific for testis.
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PMID:Identification of testis specific calcineurin beta subunit isoform by a monoclonal antibody and detection of a specific six amino acid sequence. 131 15

The effects of protein kinase C (PKC) activators on gamma-aminobutyric acidA (GABAA) receptor function were studied by two-electrode voltage-clamp in Xenopus oocytes expressing brain mRNA or subunit cDNAs and in isolated mouse brain cerebellar membrane vesicles (microsacs), using 36Cl- uptake. Both oocytes and microsacs showed transient (desensitizing) and sustained (nondesensitizing) GABAA receptor responses. In oocytes expressing brain mRNA, the PKC activator phorbol myristoyl acetate (PMA), but not the inactive analog phorbol 12-monomyristate, inhibited both transient and sustained GABA-gated chloride currents. The inhibition by PMA was concentration dependent, with an EC50 of approximately 5 nM, and resulted in a decrease in the efficacy, but not the potency, of GABA. Additionally, PMA inhibited GABA-gated chloride currents in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. The effect of PMA on recombinant receptors was significantly antagonized by PKC inhibitory peptide (PKCI). In the microsac preparation, the PKC activators (-)-7-octylindolactam V and PMA inhibited the sustained phase of 36Cl- flux without altering the transient phase. The action of PMA was blocked by kinase inhibitors and by depletion of Mg-ATP and was mimicked by protein phosphatase inhibitors. These results demonstrate that activation of PKC inhibits GABAA receptor function, and the results from the microsac experiments suggest that PKC-dependent phosphorylation preferentially inactivates a nondesensitized form or state of the receptor.
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PMID:Activation of protein kinase C selectively inhibits the gamma-aminobutyric acidA receptor: role of desensitization. 131 47

Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (beta 1) encodes a 170 amino acid protein (M(r) approximately 19.3 kDa), whereas that for the testis isoform (beta 2) contains 179 residues (M(r) approximately 20.7 kDa); these two sequences show approximately 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the beta 2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (alpha 3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.
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PMID:Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B). 132 94

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.
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PMID:Regulation of epithelial chloride channels by protein phosphatase. 171 76

Two type 2A protein phosphatases, phosphatases I (Mr = 180,000) and III (Mr = 177,000), were purified to near homogeneity from human erythrocyte cytosol. Phosphatase I was composed of alpha (34 kDa), beta (63 kDa), and delta (74 kDa) subunits in a ratio of 1:1:1. Phosphatase III comprised alpha, beta, and gamma (53 kDa) subunits in the same ratio. Heparin-Sepharose column chromatography converted most of phosphatase I and 20% of phosphatase III into alpha 1 beta 1 which were indistinguishable from phosphatase IV (Usui, H., Kinohara, N., Yoshikawa, K., Imazu, M., Imaoka, T., and Takeda, M. (1983) J. Biol. Chem. 258, 10455-10463). The catalytic subunit alpha and the beta subunit of phosphatases I, III, and IV displayed identical V8 and papain peptide maps, respectively, while the peptide maps of the alpha, beta, gamma, and delta subunits were clearly distinct. The molar ratio of phosphatases I, III, and IV in erythrocyte cytosol was estimated to be 6:1:14. Comparison of molecular activities of alpha, alpha 1 beta 1, alpha 1 beta 1 delta 1, and alpha 1 beta 1 gamma 1 revealed that beta suppressed phosphorylase and P-H2B histone phosphatase activities of alpha but stimulated the P-H1 histone phosphatase activity, and delta suppressed all the phosphatase activities of alpha 1 beta 1. The gamma subunit stimulated the P-histone phosphatase activity of alpha 1 beta 1 but inhibited the phosphorylase and P-spectrin phosphatase activities. The beta subunit increased the Mg2+ or Mn2+ requirement for P-H2B histone phosphatase activity of alpha, an effect which was counteracted by delta. The effects of heparin, H1 histone, protamine, and polylysine on the phosphorylase phosphatase activity of phosphatases I, III, IV, and alpha were described and discussed in connection with the functions of the subunits.
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PMID:Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol. 283 Dec 1

The need for nonshivering heat production, a principal function of brown adipose tissue, is accentuated in neonates. Accordingly, brown fat in the rat exhibits a very pronounced process of morphological and functional maturation perinatally, reaches a peak in its differentiation and heat-generating capacity within 1-2 weeks after birth, and undergoes involutive changes later in life. The later process of dedifferentiation can be either prevented or reversed by exposing the animals to cold ambient temperature for a prolonged period of time (cold acclimatization). The regulation of both the tissue maturation processes and the superimposed acute heat production are hormone mediated. Thus, the hormone receptor system within the adipocyte membrane and the sequence of molecular events interconnecting the initial hormonal stimulus with its final intracellular effect(s) are of considerable importance. The brown adipocytes of developing rats possess adrenoreceptors that can be pharmacologically classified as beta 1 (linked to adenylate cyclase) and alpha 2 (possibly linked to guanylate cyclase), multiple forms of cyclic nucleotide dependent and independent protein kinases, a protein kinase inhibitor, and at least two distinct phosphoprotein phosphatases associated with three phosphoprotein phosphatase modulators. The characteristics and developmental alterations of these regulatory components were studied in considerable detail by our group during the past decade. The results uncovered several target systems for ontogenic modifications of hormonal responses. Strong support was obtained for the hypothesis that protein phosphorylation and dephosphorylation is a major molecular mechanism involved in the regulation of both the brown adipocyte function and its proliferative activity during ontogenic development.
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PMID:Mechanisms of hormonal regulations in brown adipose tissue of developing rats. 614 37

A partially purified pig heart phosphoprotein phosphatase was dissociated into three distinct components, namely alpha, beta, and gamma, by gel filtration on Sephacryl S-200 followed by chromatography on DEAE-Sephadex in the presence of 6 M urea. Although alpha itself had phosphatase activities toward P-H2B histone, P-H1 histone, phosphorylase a, and glycogen synthase b, beta and gamma had no activity toward these substrates even in the presence of 1 mM Mn2+. The beta component (Mr = 80,000) combined with alpha (Mr = 31,000) in the absence of urea to produce Form 2 (Mr = 123,000) with concomitant increase in P-H1 histone phosphatase activity and Mg2+ requirement for P-H2B histone phosphatase activity (Imazu, M., Imaoka, T., Usui, H., Kinohara, N., and Takeda, M. (1981) J. Biochem. 90, 851-862). The gamma component (Mr = 62,000) reassociated with Form 2 to produce Form 1 (Mr = 199,000) which was similar to the original phosphoprotein phosphatase in substrate specificity and Mg2+ requirement. Binding of gamma to Form 2 strongly suppressed the phosphatase activities toward phosphorylase a and glycogen synthase b with marginal effects on the other phosphatase activities and Mg2+ requirement. However, gamma alone could not associate with alpha. The gamma component was sensitive to treatment with heat (60 degrees C for 2 min) or trypsin and was resistant to treatment with DNase or RNase. The pig heart phosphoprotein phosphatase was further purified to near homogeneity, as judged by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis revealed that the purified enzyme (Mr = 171,000) was composed of three polypeptide components, namely alpha', beta', and gamma' with molecular weights of 34,000, 69,000, and 56,000, respectively. The component stoichiometry was determined to be alpha' 1 beta' 1 gamma' 1 by densitometric tracing of the Coomassie blue-stained bands on the acrylamide gel. After dissociation of alpha ' and other components by gel filtration of the purified enzyme on Sephacryl S-200 in the presence of 6 M urea, one alpha ' combined with one beta' to produce Form 2' of Mr = 106,000. Since Form 1 and the purified enzyme as well as Form 2 and Form 2' had similar catalytic properties and s20,w values, respectively, component compositions are suggested to be alpha 1 beta 1 gamma 1 for Form 1 and alpha 1 beta 1 for Form Form 2.
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PMID:Resolution and reassociation of three distinct components from pig heart phosphoprotein phosphatase. 629 3

Using Chinese hamster ovary cell lysate, an in vitro assay has been developed to study the interaction of fibronectin with the alpha 5 beta 1 integrin in a cytosolic environment. In our solid phase assay, 96-well microtiter plates were coated with fibronectin in which cell lysate was incubated. A dose-dependent binding of the fibronectin receptor onto the coated plastic was immunodetected by specific polyclonal antibodies raised against the alpha 5 beta 1 integrin. Both soluble fibronectin and PB1, a monoclonal antibody raised against the fibronectin receptor, competed with the alpha 5 beta 1 integrin for binding to the fibronectin-coated plastic. General phosphatase inhibitors used during cell lysis completely abolished the fibronectin/integrin interaction in the assay, indicating that the affinity of the fibronectin receptor might be modulated by a protein phosphatase activity. Furthermore, in this assay, the interaction between the fibronectin receptor and its substrate in a cytosolic environment required intracellular calcium. Additionally, the action of more specific phosphatase inhibitors and the inhibition of the integrin/fibronectin interaction by a monoclonal antibody raised against the calcium/calmodulin-dependent protein phosphatase calcineurin suggested that calcineurin allowed the interaction between the alpha 5 beta 1 integrin and fibronectin. Metabolical labeling experiments showed that alpha 5 beta 1 itself was not the target of phosphorylation/dephosphorylation cascades involving calcineurin and leading to the modulation of integrin affinity. Taken together, these results showed that in vitro one substrate of the serine/threonine protein phosphatase calcineurin regulates the alpha 5 beta 1 integrin affinity by interacting with a yet unidentified effector.
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PMID:Control of the alpha 5 beta 1 integrin/fibronectin interaction in vitro by the serine/threonine protein phosphatase calcineurin. 753 36

Contraction of intraocular fibrous membranes is an important feature in the pathogenesis of retinal detachment in proliferative vitreoretinopathy (PVR). Collagen gel contraction is a useful in vitro model of membrane contraction in PVR. We studied the role of protein kinase C (PKC) in collagen gel contraction induced by bovine choroidal fibroblasts and retinal pigment epithelial (RPE) cells. Collagen gels embedded with the cells were formed in culture dishes and gel contraction was evaluated. The PKC stimulator, phorbol 12-myristate 13-acetate (PMA), and the protein phosphatase 1 and 2A inhibitor, okadaic acid (OA), were used to evaluate the role of the PKC-mediated phosphorylation system in this gel contraction. Fifteen min incubation with PMA stimulated gel contraction, but 180 min incubation had no effect. Choroidal fibroblast- but not RPE cell-induced gel contraction was stimulated by OA. These effects were inhibited by the broad spectrum protein kinase inhibitor staurosporine and the specific PKC antagonist calphostin C. Transforming growth factor-beta (TGF-beta)1 and TGF-beta 2, which are known to be present in eyes with PVR, were evaluated to determine their effect on gel contraction. Both TGF-beta 1 and 2 had a stimulatory effect on contraction of gels seeded with choroidal fibroblasts and RPE cells, but staurosporine and calphostin C inhibited this TGF-beta-induced gel contraction. These results indicate that activation of PKC/protein phosphorylation is an important factor in gel contraction caused by choroidal fibroblasts and RPE cells, and that TGF-beta-induced gel contraction is mediated at least in part via the PKC pathway.
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PMID:Collagen gel contraction induced by retinal pigment epithelial cells and choroidal fibroblasts involves the protein kinase C pathway. 792 9

Using both a protein phosphatase inhibitor, okadaic acid (OA), and a protein kinase inhibitor, H7, to modify phosphorylation events in the cell, we investigated the effects of these agents on transcriptional activation via exogenous rat thyroid hormone receptor (TR) isoforms in transiently transfected cells and endogenous TRs. CV-1 cells were transiently cotransfected with expression plasmids encoding either the rat TR alpha 1 or TR beta 1, and luciferase reporter plasmids containing either the synthetic DR4 or the chick lysozyme F2 thyroid hormone response elements (TREs). For both receptor isoforms, there was an enhancement of transcriptional activity after incubation with 5 nM T3 for 24 h compared to hypothyroid levels. There was little change in transcriptional activation in the presence of 25 nM OA alone; however, for both TR isoforms and both TREs studied, OA augmented the stimulatory effects of T3. For the F2 TRE, transcriptional activation via TR alpha 1 increased from 19- to 35-fold, and that via TR beta 1 increased from 6- to 10-fold in the presence of T3 and OA compared to that with T3 alone. Similar results were found for the DR4 TRE. OA enhanced transcriptional activation by T3 in a dose-dependent manner. Increasing concentrations of OA (0, 5, 25, and 50 nM) further increased relative luciferase activity from 11-fold in the absence of OA to 45-fold in the presence of 50 nM OA. The protein kinase inhibitor, H7, caused no change in the transcriptional activity of the reporter plasmids via TR alpha 1 in the absence of T3, but completely blocked transcriptional activation by T3 for both the DR4 and the F2 TREs. H7 also blocked stimulation of endogenous GH and inhibition of endogenous TR beta 2 mRNAs by T3 in GH3 cells. These results indicate that phosphorylation events in the cell play an important role in transcriptional activation via both TR isoforms.
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PMID:Evidence that phosphorylation events participate in thyroid hormone action. 829 53


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