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 effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.
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PMID:Effect of thrombin on phosphorylation of platelet membrane proteins. 98 70

The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C4 plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark-form carboxylase was accompanied by an increase in the apparent Ki of the feedback inhibitor L-malate and an increase in Vmax. Feeding detached leaves various photosynthetic inhibitors, i.e. 3-(3,4-dichlorophenyl)-1,1-dimethylurea, gramicidin and DL-glyceraldehyde, prevented PPrv carboxylase phosphorylation in the light, thus suggesting that the cascade involves the photosynthetic apparatus as the light signal receptor, and presumably has the electron transfer chain and the Calvin-Benson cycle as components in the signal-transduction chain. Two protein-serine kinases capable of phosphorylating PPrv carboxylase in vitro have been partially purified from light-adapted leaves. One was isolated on a calmodulin-Sepharose column; it was calcium-dependent but did not require calmodulin for activity. The other was purified on a blue-dextran-agarose column and the only Me2+ required for activity was Mg2+. In reconstituted phosphorylation assays, only the latter caused the expected decrease in malate sensitivity of PPrv carboxylase suggesting that this protein is the genuine PPrv-carboxylase-kinase. Desalted extracts from light-adapted leaves possessed a considerably greater phosphorylation capacity with immunopurified dephosphorylated PPrv carboxylase as substrate than did dark extracts. This light stimulation was insensitive to type 2A protein phosphatase inhibitors, okadaic acid and microcystin-LR, which suggests that the kinase is a controlled step in the cascade which leads to phosphorylation of PPrv carboxylase. The higher phosphorylation capacity of light-adapted leaf tissue was nullified by pretreatment with the cytosolic protein synthesis inhibitor, cycloheximide. Thus, protein turnover is involved as part of the mechanism controlling the activity of the kinase purified on blue-dextran-agarose. However, no information is available with respect to the specific nature of the link between the above-mentioned light transducing steps and the protein kinase that achieves the physiological response. Finally, the in vivo phosphorylation site (Ser8) in the N-terminal region of the C4 type Sorghum PPrv carboxylase is also present in a non-photosynthetic form of the Sorghum enzyme (Ser7), as deduced by cDNA sequence analysis.
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PMID:Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase. Identification of the protein-serine kinase and some elements of the signal-transduction cascade. 131 81

The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.
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PMID:Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity. 132 19

GCN2 is a protein kinase in Saccharomyces cerevisiae that is required for increased expression of the transcriptional activator GCN4 in amino acid-starved cells. GCN2 stimulates GCN4 synthesis at the translational level by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2). We identified a truncated form of the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by its ability to restore derepression of GCN4 expression in a strain containing the partially defective gcn2-507 allele. Genetic analysis suggests that the truncated GLC7 allele has a dominant negative phenotype, reducing the level of native type 1 protein phosphatase activity in the cell. The truncated form of GLC7 does not suppress the regulatory defect associated with a gcn2 deletion or a mutation in the phosphorylation site of eIF-2 alpha (Ser-51). In addition, the presence of multiple copies of wild-type GLC7 impairs the derepression of GCN4 that occurs in response to amino acid starvation or dominant-activating mutations in GCN2. These findings suggest that the phosphatase activity of GLC7 acts in opposition to the kinase activity of GCN2 in modulating the level of eIF-2 alpha phosphorylation and the translational efficiency of GCN4 mRNA. This conclusion is supported by biochemical studies showing that the truncated GLC7 allele increases the level of eIF-2 alpha phosphorylation in the gcn2-507 mutant to a level approaching that seen in wild-type cells under starvation conditions. The truncated GLC7 allele also leads to reduced glycogen accumulation, indicating that this protein phosphatase is involved in regulating diverse metabolic pathways in yeast cells.
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PMID:Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2. 133 44

During anaerobic growth, nitrate induces synthesis of the anaerobic respiratory enzymes formate dehydrogenase-N and nitrate reductase. This induction is mediated by a transcription activator, the narL gene product. The narX gene product may be involved in sensing nitrate and phosphorylating NARL. We isolated narX mutants, designated narX*, that caused nitrate-independent expression of the formate dehydrogenase-N and nitrate reductase structural genes. We used lambda narX specialized transducing phage to genetically analyze these lesions in single copy. Two previously isolated narX* mutations, narX32 and narX71, were also constructed by site-specific mutagenesis. We found that each of these alleles caused nitrate-independent synthesis of formate dehydrogenase-N and nitrate reductase, and each was recessive to narX+. The narX* mutations lie in a region of similarity with the methyl-accepting chemotaxis protein Tsr. We suggest that the narX* proteins have lost a transmembrane signalling function such that phosphoprotein phosphatase activity is reduced relative to protein kinase activity.
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PMID:Mutational analysis reveals functional similarity between NARX, a nitrate sensor in Escherichia coli K-12, and the methyl-accepting chemotaxis proteins. 159 21

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

The most prominent tyrosyl-phosphorylated protein in maturing sea star oocytes was identified as the 44 kDa myelin basic protein (MBP) kinase p44mpk. Immunoblotting studies with anti-phosphotyrosine PY-20 antibody and phosphoamino acid analysis of in vivo [32P]phosphate-labelled p44mpk showed that the tyrosyl phosphorylation of the kinase correlated with a greater than 10-fold stimulation of its MBP phosphotransferase activity. The activation of p44mpk was reversed almost completely by purified preparations of the protein-tyrosyl phosphatases CD45 and 1B. Purified p44mpk has previously been shown to undergo autophosphorylation in vitro on seryl residues and this was associated with further enhancement of its MBP phosphorylating activity (Sanghera et al. (1991) J. Biol. Chem. 266, 6700-6707). p44mpk also underwent seryl phosphorylation during oocyte maturation, and the protein-seryl/threonyl phosphatase 2A reversed partially the maturation-associated stimulation of its MBP kinase activity. The properties of p44mpk resemble the murine 42 kDa mitogen-activated protein kinase (p42mapk). While p44mpk may feature the phosphorylatable tyrosyl residue that is critical for activation in p42mapk, it lacks the upstream threonyl phosphorylation site that is also required for p42mapk activity (Payne et al. (1991) EMBO J: 10, 885-892). These findings indicate partial differences in the regulatory mechanisms that govern the activities of these isozymes.
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PMID:Tyrosyl phosphorylation and activation of the myelin basic protein kinase p44mpk during sea star oocyte maturation. 171 36

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.
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PMID:Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system. 185 Mar 5

Activity of crude histidine decarboxylases (HisDC) from the hypothalamus and the lungs, was markedly reduced by incubating with ATP.Mg, cAMP and cAMP-dependent protein kinase A, whereas activity of the crude glandular stomach enzyme changed only slightly under equal condition. The omission of one of these components failed to reduce HisDC activity by as much as the complete system. Addition of bovine heart (type II) or rat cerebellum protein kinase A (types I and II) inhibitor to the assay prevented enzyme inactivation; moreover, protein kinase A inhibitors permitted moderate activation under phosphorylating and control conditions. Cytosolic hypothalamus HisDC activity was elevated 2-2.2-fold by incubating the cytosol for 15 min in the presence of MnCl2, a known stimulator of phosphoprotein phosphatase; this was prevented when 20 mM NaF, a common inhibitor of phosphoprotein phosphatase, was added to the cytosol. The apparent Km of ATP.Mg-treated hypothalamus HisDC for histidine was elevated 5-10-fold compared to controls, whereas the Vmax was approximately the same. Under this condition, the Km was calculated as high as 0.5-2.2 mM (depending on phosphorylating conditions), while controls had a Km of 0.1-0.3 mM (depending on the initial phosphorylating states). Addition of rabbit muscle (type I), bovine heart (type II) or rat cerebellum (types I and II) inhibitor of protein kinase A, to the phosphorylating mixture, abolished the difference in Km between control and ATP.Mg-treated HisDC. Moreover, rat cerebellum protein kinase A inhibitors increased Vmax to above the control level; while 20 mM NaF (inhibitor of phosphoprotein phosphatase) decreased Vmax to approximately one half of that of the controls. These data indicate that HisDC activity in the hypothalamus and the lungs, but not in the stomach, is affected in oppositely by protein kinase A and phosphoprotein phosphatases.
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PMID:Possible regulation of hypothalamus and lung histidine decarboxylase activity by cAMP-dependent protein kinase. 201 19

The dominant insulin-stimulated ribosomal protein S6 kinase activity was purified to near homogeneity from insulin-treated 32P-labeled rat H4 hepatoma cells and found to copurify with a 70-kDa 32P-labeled polypeptide. The dominant S6 kinase purified from livers of cycloheximide-treated rats is also a 70-kDa polypeptide. Antiserum raised against rat liver S6 kinase specifically immunoprecipitates the purified 32P-labeled H4 hepatoma insulin-stimulated S6 kinase. This antiserum also specifically precipitates insulin-stimulated S6 kinase activity directly from cytosolic extracts of H4 cells. Immune complexes prepared from the cytosol of 32P-labeled H4 cells contain several 32P-labeled polypeptides; only a 70-kDA 32P-labeled peptide, however, is specifically displaced by preadsorption of the antiserum with nonradioactive rat liver S6 kinase. Insulin treatment increases the 32P content of the immunoprecipitated 70-kDa S6 kinase polypeptide 3- to 4-fold over basal levels; 32P-labeled serine, some 32P-labeled threonine, but no 32P-labeled tyrosine are detected after partial acid hydrolysis. Tryptic peptide maps indicate that the insulin-stimulated S6 kinase purified from 32P-labeled H4 cells is phosphorylated at multiple sites distinct from those which participate in autophosphorylation in vitro. Autophosphorylation of rat liver S6 kinase in vitro does not modify S6 kinase activity. The S6 kinases purified from liver of cycloheximide-treated rat and H4 hepatoma insulin-stimulated enzyme are each completely deactivated by incubation with protein phosphatase type 2A in both autophosphorylating and 40S S6 phosphorylating activities. The phosphatase 2A-deactivated 70-kDa S6 kinase is neither reactivated nor phosphorylated by partially purified insulin-stimulated microtubule-associated protein 2 kinase, in experiments where Xenopus S6 kinase II undergoes phosphorylation and partial reactivation. Thus insulin activates the 70-kDa S6 kinase by promoting phosphorylation of specific serine/threonine residues on the enzyme polypeptide, probably through activating an as-yet-unidentified serine/threonine protein kinase distinct from microtubule-associated protein 2 kinase.
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PMID:Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide. 212 50


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