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)

Calcineurin, a calmodulin-stimulated protein phosphatase, was a substrate for purified bovine brain protein carboxyl O-methyltransferase (protein O-methyltransferase; EC 2.1.1.24) and incorporated up to 2 mol of CH3 per mol of calcineurin. Carboxyl methylation was dependent on the concentrations of S-adenosyl-L-[methyl-3H]methionine and was prevented by addition of the carboxyl methylation inhibitor S-adenosylhomocysteine. The stoichiometry of methyl group incorporation was related to the ratio of methyltransferase/calcineurin. The rate of spontaneous hydrolysis of carboxyl methylester groups on calcineurin increased rapidly above pH 6.5 with those on native carboxyl-methylated calcineurin substantially more labile than for trichloracetic acid-precipitated calcineurin. Polyacrylamide gel electrophoresis in the presence of NaDodSO4 (pH 2.4) confirmed that the A subunit of calcineurin (Mr = 61,000) was the primary site of carboxyl methylation with little, if any, modification of the B subunit (Mr = 18,000). When carboxyl-methylated calcineurin (approximately 1-2 mol of CH3 per mol of protein) was assayed for p-nitrophenyl phosphatase activity at pH 6.5, a marked inhibition of calmodulin-stimulated activity was observed while there was little effect on Mn2+-stimulated phosphatase activity. Thus, calcineurin appears to be an excellent substrate for protein carboxyl O-methylation and this modification, which impairs calmodulin stimulation of phosphatase activity, may be of functional significance.
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PMID:Stoichiometric methylation of calcineurin by protein carboxyl O-methyltransferase and its effects on calmodulin-stimulated phosphatase activity. 299 37

The enzymes protein-carboxyl methylase and protein methylesterase reversibly modify the charge and structure of proteins by adding and removing methyl groups on free carboxyl groups of proteins. Since this posttranslational system has been implicated in biological processes that required calcium, the carboxyl methylation of calmodulin was investigated. Calmodulin was an excellent substrate for both protein-carboxyl methylase and protein methylesterase. Carboxyl methylation of calmodulin resulted in inactivation, since methylated calmodulin was less capable of activating cyclic nucleotide phosphodiesterase. To determine whether the carboxyl methylation of calmodulin was simply a test tube reaction or a biochemical reaction normally occurring in intact cells, two different cell lines were labeled with [methyl-3H]methionine. Calmodulin was isolated by affinity chromatography and was found to be carboxyl methylated. Finally, calcineurin was also an excellent substrate for the methylase, suggesting that other calcium-binding proteins may be affected by protein methylation-demethylation.
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PMID:Enzymatic carboxyl methylation of calcium-binding proteins. 631 68

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a major endogenous cytosolic substrate for dopamine- and cyclic AMP-stimulated protein phosphorylation in neurons of the basal ganglia of mammalian brain. It shares many properties with phosphatase inhibitor 1, a substrate for cyclic AMP-dependent protein kinase, and with G-substrate, a substrate for cyclic GMP-dependent protein kinase. We have, therefore, undertaken an analysis of the amino acid sequence around the site at which purified DARPP-32 is phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. The results indicate that DARPP-32 is phosphorylated at a single threonine residue contained in the sequence Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Met-Leu-Phe-Arg. This sequence was obtained by automated solid phase sequencing of two overlapping tryptic phosphopeptides and one overlapping chymotryptic phosphopeptide which were purified by reverse-phase high-performance liquid chromatography. A 9-amino acid sequence containing the phosphorylatable threonine residue in DARPP-32 shares 8 identical residues with a sequence containing the phosphorylatable threonine residue in phosphatase inhibitor 1, and shares 5 identical residues with the two identical sequences surrounding the 2 phosphorylatable threonine residues in G-substrate. These observations support the view that DARPP-32, inhibitor 1, and G-substrate are members of a family of regulatory proteins which are involved in the control of protein phosphatase activity by both cyclic AMP and cyclic GMP, but which differ in their cellular and tissue distributions.
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PMID:DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine. 650 2

Human UC11 astrocytoma cells were used to investigate the role of protein kinase C (PKC) and other kinases in neurokinin (NK)1 receptor desensitization. The selective NK1 receptor agonist [Sar9,Met(O2)11]-substance P stimulated a biphasic accumulation of [3H]inositol phosphates ([3H]IPs) in the presence of 10 mM LiCl in cells that had been prelabeled with [3H]inositol. An initial rapid phase of [3H]IP accumulation during the first 1 min was followed by a slower sustained phase for up to 90 min. These results demonstrate that the human NK1 receptor desensitizes rapidly but only partially. The selective PKC inhibitor Ro31-8220 did not prevent rapid NK1 receptor desensitization but after a longer incubation significantly potentiated human NK1 receptor agonist-stimulated accumulation of [3H]IPs. These results suggest that, although PKC does not mediate the process of rapid desensitization, it does have an inhibitory role at later times. This conclusion is supported by studies with staurosporine, phorbol dibutyrate, and the protein phosphatase inhibitor okadaic acid. Studies using AlF4-, an agent that can directly activate G proteins, and Ro31-8220 suggested that PKC can exert inhibitory effects 'downstream' of receptor activation, although immunoprecipitation of the G proteins alpha q/alpha 11 demonstrated that they do not undergo phosphorylation in UC11 cells and are unlikely to be the target of PKC-mediated inhibitory feedback. Delayed inhibitory feedback by PKC may be mediated by phosphorylation of phospholipase C, although an additional site of action on the NK1 receptor cannot be ruled out.
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PMID:Protein kinase C mediates delayed inhibitory feedback regulation of human neurokinin type 1 receptor activation of phospholipase C in UC11 astrocytoma cells. 752 12

Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells.
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PMID:Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A. 753 75

Cytosolic components of the phagocyte NADPH oxidase (p47phox, p67phox, and Rac2) translocate to the plasma membrane on cell activation where they interact with a membrane-bound cytochrome b to generate superoxide anion. Phosphorylation reactions are known to be important for activity of NADPH oxidase. Translocation of Rac2, p47phox, and p67phox were all enhanced in formyl-Met-Leu-Phe-stimulated neutrophils treated with 50 nM of the protein phosphatase 1/2A inhibitor calyculin A. Rac translocation was blocked by the tyrosine kinase inhibitors genistein (50 microM) and herbimycin (17 microM), whereas movement of p47phox and p67phox were not inhibited. Cell-free analysis of Rac translocation also demonstrated that translocation of p47phox and p67phox were not linked to the movement or availability of Rac2. Thus, Rac2 does not appear to regulate NADPH oxidase by controlling movements of the cytosolic components to the membrane-associated enzyme but may exert its effect at the level of the assembled complex. Tyrosine kinase activity is required for translocation of Rac in the chemoattractant-stimulated human neutrophil.
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PMID:Dissociation of Rac translocation from p47phox/p67phox movements in human neutrophils by tyrosine kinase inhibitors. 761 2

A cDNA clone encoding the calcium-binding subunit of calcineurin, calcineurin B, was isolated from a bovine brain library by immunoscreening. The 841 bp cDNA has a 56 bp 5'-noncoding region, an open reading frame of 510 bp, and a 275 bp 3'-noncoding sequence. The deduced amino acid sequence of bovine calcineurin B differs from the previously reported protein sequence (Aitken et al., 1984) by three residues. The sequence contained additional valine at the carboxyl terminus and substitutions of Met-11 and Ser-153 (the positions according to Aitken et al., 1984) by cysteine. The amino acid sequence of bovine calcineurin B was found to be identical to that of human calcineurin B sequence (Guerini et al., 1989). In fact, 97.1% homology was observed between the coding regions of human and bovine calcineurin B. In addition, a very high homology of 95.2% was observed for the 3'-noncoding region while the 5'-noncoding region showed 58.9% homology. The beta-galactosidase fusion protein, having the apparent molecular weight of 29 kDa, was detected on Western blots by subunit B-specific monoclonal antibody (Matsui et al., 1985). Northern analysis revealed that there is a single calcineurin B transcript in bovine brain which is 2.3 kb in length. This is in agreement with the observation of only one immunologically detectable subunit B protein in bovine brain (Matsui et al., 1985).
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PMID:Isolation and characterization of a cDNA clone coding for the calcium-binding subunit of calcineurin from bovine brain: an identical amino acid sequence to the human protein. 780 16

The peptide neurotransmitter Phe-Met-Arg-PheNH2 (FMRFamide) increases outward K+ currents and promotes dephosphorylation of many phosphoproteins in Aplysia sensory neurons. We examined FMRFamide-induced current responses in sensory neurons injected with thiophosphorylated protein phosphate inhibitor-1 and inhibitor-2 (I-1 and I-2), two structurally different vertebrate protein phosphatase-1 (PP1) inhibitors to define a role for PP1 in the physiological actions of FMRFamide. Thiophosphorylated I-1 and I-2 both reduced the amplitude of outward currents elicited by FMRFamide by 50-60% and were as effective as microcystin-LR, which inhibited both PP1 and protein phosphatase-2A in Aplysia neuronal extracts. These data suggested that of the two major neuronal protein serine/threonine phosphatases, FMRFamide utilized primarily PP1 to open serotonin-sensitive K+ (S-K+) channels. Earlier studies showed that a membrane-associated phosphatase regulated S-K+ channels in cell-free patches from sensory neurons. Utilizing its unique substrate specificity and inhibitor sensitivity, we have characterized PP1 as the principal protein phosphatase associated with neuronal plasma membranes. Two protein phosphatase activities (apparent M(r) values of 170,000 and 38,000) extracted from crude membrane preparations from the Aplysia nervous system were shown to be isoforms of PP1. These biochemical and physiological studies suggest that PP1 is preferentially associated with neuronal membranes and that its activity may be required for the induction of outward K+ currents in the Aplysia sensory neurons by FMRFamide.
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PMID:Protein phosphatase-1 regulates outward K+ currents in sensory neurons of Aplysia californica. 789 Nov 12

A clone encoding the catalytic subunit of a protein phosphatase from Saccharomyces cerevisiae was isolated. Except for replacement of IIe-245 by Met the structure of the phosphatase was identical to that encoded by PPH3 (Ronne, H., Carlberg, M., Hu, G. Z. and Nehlin, J. O. (1991). Mol. Cell. Biochem. 11, 4876-4884) and exhibited 63% sequence identity to PPX cloned from a rabbit liver cDNA library (Brewis, N.D., Street, A.J., Prescott, A.R. and Cohen, P.T.W. (1993). EMBO J. 12, 987-996). Expression of active enzyme was achieved in Escherichia coli mutants which were generated by a genetic selection based on functional complementation of bacterial phosphoserine phosphatase. Though some of the properties of PPH3 resembled those of protein phosphatase 2A and PPX, others were different. PPH3 exhibited lower sensitivity against inhibition by okadaic acid, showed different substrate specificity and required a divalent cation (Mn2+ was preferred before Mg2+ and Ca2+) for activity when assayed with phospho-histone as a substrate. However, 25% of maximum activity was observed in the absence of divalent cations when the peptide LRRAS(P)LG was used as substrate. The PPH3-protein was also identified by chromatography of extracts from S. cerevisiae on DEAE-cellulose. Protein immunoreactive with an antiserum raised against the non-conserved N-terminal 53 amino acids of PPH3 was coeluted with a single peak of LRRAS(P)LG dephosphorylating activity.
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PMID:The Saccharomyces cerevisiae gene PPH3 encodes a protein phosphatase with properties different from PPX, PP1 and PP2A. 794 42

Initiation factor eIF-2 (a trimer of subunits alpha, beta and gamma) attaches the initiator Met-tRNA to the ribosome during the initiation of translation in eukaryotic cells. Both the alpha and beta subunits can be phosphorylated although the sites in the beta-subunit have not previously been fully identified. Here we identify the sites at which eIF-2 beta is phosphorylated in vitro by three well-characterised protein kinases, casein kinase-2 (which phosphorylates serine residues-2 and -67), protein kinase C (serine-13) and cAMP-dependent protein kinase (serine-218). This constitutes an essential prerequisite for studying the phosphorylation of eIF-2 beta in vivo. Indeed, we present evidence that at least one of these sites (serine-67) is phosphorylated in reticulocytes. The major kinase activity against eIF-2 beta in reticulocyte lysates appears in CK-2 and protein phosphatase-2A is the principal enzyme responsible for dephosphorylation of eIF-2 beta phosphorylated by this kinase.
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PMID:Identification of novel phosphorylation sites in the beta-subunit of translation initiation factor eIF-2. 802 72


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