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 synthetic phosphopeptide RRATpVA was found to be the most effective substrate for protein phosphatase 2C (PP2C) so far identified. Replacement of phosphothreonine by phosphoserine decreased activity over 20-fold and a striking preference for phosphothreonine was also observed with two other substrates (RRSTpTpVA and casein) that were phosphorylated on both serine and threonine. Replacement of the C-terminal valine in RRATpVA by proline abolished dephosphorylation, while exchanging the N-terminal alanine by proline had no effect. The preference for phosphothreonine and the effect of proline are similar to protein phosphatase 2A (PP2A). However, the peptide RRREEETpEEEAA, an excellent substrate for PP2A, was not dephosphorylated by PP2C, and substitution of the C-terminal valine in RRATpVA by glutamic acid reduced the rate of dephosphorylation by PP2C over 10-fold, without affecting dephosphorylation by PP2A. Addition of two extra N-terminal arginine residues to RRASpVA increased PP2A catalysed dephosphorylation 4- to 5-fold, without altering dephosphorylation by PP2C. These results represent the first study of the specificity of PP2C using synthetic peptides, and strengthen the view that this approach may lead to the development of more effective and specific substrates for the serine/threonine-specific protein phosphatases.
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PMID:An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A. 215 67

We have identified and cloned human cDNA for the Ca2+-binding subunit of calcineurin, the brain isozyme of the Ca2+/calmodulin-stimulated protein phosphatase. The 2.5-kb cDNA has an open reading frame of 510 bp, a leader sequence of at least 500 bp, and a 1,277-bp 3'-noncoding sequence. The deduced sequence of the human protein differs from bovine brain calcineurin B by an additional valine at the carboxyl terminus and substitution of Met-11 and Ser-153 by cysteine. A partial clone of the mouse protein corresponding to amino acids 75-150 was also isolated. This portion of the human and mouse protein sequence is identical, with the DNA sequences showing 94% identity. The respective mRNAs in human and mouse are also of similar size. As was observed with protein levels, mRNA abundance in brain is 20-60 times that found in other tissues with the exception of HeLa cells which, like brain, contain abundant calcineurin B mRNA.
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PMID:Isolation and sequence of a cDNA clone for human calcineurin B, the Ca2+-binding subunit of the Ca2+/calmodulin-stimulated protein phosphatase. 255 68

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 activity of Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) is not regulated by phosphorylation, presumably due to the absence of a suitable target serine and protein kinase recognition motif. We have engineered P. mevalonii HMG-CoA reductase to a form whose activity, like that of mammalian HMG-CoA reductases, is regulated by phosphorylation/dephosphorylation. We substituted serine for arginine 387, the residue that corresponds to the regulatory serine of the HMG-CoA reductases of higher eukaryotes. A recognition motif for cAMP-dependent protein kinase was added by replacing leucine 384 by histidine (enzyme L384H/R387S) and also valine 391 by leucine (enzyme L384H/R387S/V391L). The activity of P. mevalonii HMG-CoA reductase mutant enzymes L384H/R387S and L384H/R387S/V391L was attenuated by phosphorylation. Restoration of activity accompanied subsequent dephosphorylation catalyzed by lambda protein phosphatase. Incorporation and subsequent release of phosphate paralleled the attenuation and restoration of catalytic activity. Incorporation of 0.5 mol of phosphate per subunit was accompanied by an approximately 50% decrease in initial activity. As in the analogous Syrian hamster mutant enzyme S871D, P. mevalonii mutant enzyme R387D exhibited 10% wild-type activity, suggesting that the attenuation of activity that accompanies phosphorylation results at least in part from the introduction of negative charge. Engineering of P. mevalonii HMG-CoA reductase to forms whose activity is reversibly regulated by phosphorylation/dephosphorylation provides an attractive model for future structure-based mechanistic studies. Solution of the X-ray structure of phosphorylated and dephosphorylated forms of engineered P. mevalonii HMG-CoA reductase should then reveal interactions of the active site phosphoseryl residue that result in attenuation of catalytic activity.
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PMID:Protein engineering of the HMG-CoA reductase of Pseudomonas mevalonii. Construction of mutant enzymes whose activity is regulated by phosphorylation and dephosphorylation. 904 17

The calcium/calmodulin-dependent activation of nitric-oxide synthase (NOS) and its production of nitric oxide (NO) play a key regulatory role in plant and animal cell function. SCaM-1 is a plant calmodulin (CaM) isoform that is 91% identical to mammalian CaM (wild type CaM (wtCaM)) and a selective competitive antagonist of NOS (Cho, M. J., Vaghy, P. L., Kondo, R., Lee, S. H., Davis, J. P., Rehl, R., Heo, W. D., and Johnson, J. D. (1998) Biochemistry 37, 15593-15597). We have used site-directed mutagenesis to show that a point mutation, involving the substitution of valine for methionine at position 144, is responsible for SCaM-1's inhibition of mammalian NOS. An M144V mutation in wild type CaM produced a mutant (M144V) which exhibited nearly identical inhibition of NOS's NO production and NADPH oxidation, with a similar K(i) (approximately 15 nM) as SCaM-1. A V144M back mutation in SCaM-1 significantly restored its ability to activate NOS's catalytic functions. The length of the hydrophobic amino acid side chain at position 144 appears to be critical for NOS activation, since M144L and M144F activated NOS while M144V and M144C did not. Despite their competitive antagonism of NOS, M144V, like SCaM-1, exhibited a similar dose-dependent activation of phosphodiesterase and calcineurin as wtCaM. SCaM-1 and M144V produced greater inhibition of NOS's oxygenase domain function (NO production) than its reductase domain functions (NADPH oxidation and cytochrome c reduction). Thus, CaM's methionine 144 plays a critical role the activation of NOS, presumably by influencing the function of NOS's oxygenase domain.
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PMID:A point mutation in a plant calmodulin is responsible for its inhibition of nitric-oxide synthase. 1059 8

Cyclosporin A (CsA) shows cytoprotective properties in many cellular and in vivo models that may depend on interference of the interaction of cyclophilin A with calcineurin or of cyclophilin D with the mitochondrial permeability transition (PT) pore. The nonimmunosuppressive cyclosporin derivative N-methyl-4-valine-cyclosporin (PKF220-384) inhibits the mitochondrial permeability transition (MPT) like CsA but without calcineurin inactivation. PKF220-384 has been used to discriminate between PT pore- and calcineurin mediated effects but is no longer available. Here, we evaluated the effects of another nonimmunosuppressive cyclosporin derivative, N-methyl-4-isoleucine-cyclosporin (NIM811) on the MPT. Using two newly developed microtiter plate assays, one measuring mitochondrial swelling from absorbance and the other measuring mitochondrial membrane potential from changes in safranin fluorescence, we show that NIM811 blocks the MPT induced by calcium and inorganic phosphate, alone or in combination with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the complex I inhibitor rotenone, and the prooxidant t-butylhydroperoxide. NIM811 was equipotent to CsA and half as potent as PKF220-384. Additionally, we show that NIM811 blocks cell killing and prevents in situ mitochondrial inner membrane permeabilization and depolarization during tumor necrosis factor-alpha-induced apoptosis to cultured rat hepatocytes. NIM811 inhibition of apoptosis was equipotent with CsA except at higher concentrations: CsA lost efficacy but NIM 811 did not. We conclude that NIM811 is a useful alternative to PKF220-384 to investigate the role of the mitochondrial permeability transition in apoptotic and necrotic cell death.
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PMID:Inhibition of the mitochondrial permeability transition by the nonimmunosuppressive cyclosporin derivative NIM811. 1206 51

Previous work from our laboratory and others has established that Ste-20-related proline-alanine-rich kinase (SPAK/PASK) is central to the regulation of NKCC1 function. With no lysine (K) kinase (WNK4) has also been implicated in the regulation of NKCC1 activity through upstream activation of SPAK. Because previous studies from our laboratory also demonstrated a protein-protein interaction between SPAK and apoptosis-associated tyrosine kinase (AATYK), we explore here the possibility that AATYK is another component of the regulation of NKCC1. Heterologous expression of AATYK1 in NKCC1-injected Xenopus laevis oocytes markedly inhibited cotransporter activity under isosmotic conditions. Interestingly, mutation of key residues in the catalytic domain of AATYK1 revealed that the kinase activity does not play a role in the suppression of NKCC1 function. However, mutagenesis of the two SPAK-binding motifs in AATYK1 completely abrogated this effect. As protein phosphatase 1 (PP1) also plays a central role in the dephosphorylation and inactivation of NKCC1, we investigated the possibility that AATYK1 interacts with the phosphatase. We identified a PP1 docking motif in AATYK1 and demonstrated interaction using yeast-2-hybrid analysis. Mutation of a key valine residue (V1175) within this motif prevented protein-protein interaction. Furthermore, the physical interaction between PP1 and AATYK was required for inhibition of NKCC1 activity in Xenopus laevis oocytes. Taken together, our data are consistent with AATYK1 indirectly inhibiting the SPAK/WNK4 activation of the cotransporter by scaffolding an inhibitory phosphatase in proximity to a stimulatory kinase.
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PMID:Apoptosis-associated tyrosine kinase scaffolding of protein phosphatase 1 and SPAK reveals a novel pathway for Na-K-2C1 cotransporter regulation. 1726 45

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.
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PMID:Effect of branched-chain amino acids on muscle atrophy in cancer cachexia. 1762 10

Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with a group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in cis with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus, competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca(2+)-calcineurin signaling.
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PMID:The RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators. 1933 97

Modulation of the expression of the protein phosphatase-1 (PP1) glycogen-targeting subunit PTG exerts profound effects on cellular glycogen metabolism in vitro and in vivo. PTG contains three distinct binding domains for glycogen, PP1, and a common site for glycogen synthase and phosphorylase. The impact of disrupting the PP1-binding domain on PTG function was examined in 3T3-L1 adipocytes. A full-length PTG mutant was generated as an adenoviral construct in which the valine and phenylalanine residues in the conserved PP1-binding domain were mutated to alanine (PTG-VF). Infection of fully differentiated 3T3-L1 adipocytes with the PTG-VF adenovirus reduced glycogen stores by over 50%. In vitro, PTG-VF competitively interfered with wild-type PTG action, suggesting that the mutant construct acted as a dominant-negative molecule. The reduction in cellular glycogen storage was due to a significantly increased rate of glycogen turnover. Interestingly, acute basal and insulin-stimulated glucose uptake and glycogen synthesis rates were enhanced in PTG-VF expressing cells vs. control 3T3-L1 adipocytes, likely as a compensatory response to the loss of glycogen stores. These results indicate that the mutation of the PP1-binding domain on PTG resulted in the generation of a dominant-negative molecule that impeded endogenous PTG action and reduced cellular glycogen levels, through enhancement of glycogenolysis rather than impairment of glycogen synthesis.
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PMID:Generation of a dominant-negative glycogen targeting subunit for protein phosphatase-1. 2020 31


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