Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.
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PMID:Inhibition of neuronal nitric-oxide synthase by calcium/ calmodulin-dependent protein kinase IIalpha through Ser847 phosphorylation in NG108-15 neuronal cells. 1087 31

The G(M) glycogen-targeting subunit of protein phosphatase-1 (PP1) is believed to be involved in dephosphorylation of the enzymes of glycogen metabolism. To assess the roles of G(M) on glycogen metabolism, we created site-directed G(M) mutants and overexpressed them in Chinese hamster ovary (CHO) cells expressing human insulin receptor. Overexpressed G(M) recruited glycogen synthase as well as PP1 to the glycogen pellet, and upregulated basal glycogen synthase activity. Overexpressed G(M)-67A (Ser-67 replaced with alanine) exhibited decreased sensitivity to suppression of glycogen synthase activity by forskolin, while overexpression of G(M)-48A (Ser-48 replaced with alanine) preserved glycogen synthase activation in response to insulin. These observations indicate that in CHO cells overexpressing G(M); (1) G(M) translocates glycogen synthase to the glycogen pellet and affected basal glycogen synthase, (2) Ser-67 might be involved in the suppression of glycogen synthase activity by glycogenolytic agents, and (3) Ser-48 might not commit to activation of glycogen synthase by insulin.
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PMID:Overexpression of the glycogen targeting (G(M)) subunit of protein phosphatase-1. 1097 12

Protein phosphatase 2A is ubiquitous among eukaryotes and exists as a family of holoenzymes in which the catalytic subunit. PP2Ac, binds a variety of regulatory subunits. Using the yeast Saccharomyces cerevisia, we have investigated the role of the phylogenetically invariant C-terminal leucine residue of PP2Ac, which, in mammalian cells, undergoes reversible methylation and modulates binding of the PR55/B subunit. In S. cerevisiae, the C-terminal Leu-377 residue of Pph22p (equivalent to human PP2Ac Leu-309) was dispensable for cell growth under optimum conditions and its removal, or substitution by alanine, did not inhibit PP2A activity in vitro. However, Leu-377 is required for binding of the yeast PR55/B subunit, Cdc55p, by Pph22p, though apparently not for the binding of Rts1p, the yeast PR61/B' subunit. Furthermore, mutation of this leucine enhanced the sensitivity of cells to microtubule destabilization, a defect characteristic of cdc55delta mutant cells, which are impaired for spindle checkpoint function. These results demonstrate that the regulation of PP2A, mediated by PR55/B binding to the highly conserved PP2Ac C-terminus, is critical for cell viability under conditions of microtubule damage and support a role for PP2A in exit from mitosis.
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PMID:Mutation of the C-terminal leucine residue of PP2Ac inhibits PR55/B subunit binding and confers supersensitivity to microtubule destabilization in Saccharomyces cerevisiae. 1112 46

The catalytic subunit of the Ser/Thr protein phosphatase 1 (PP1cat) hydrolyses N-acetyl Arg-Arg-Ala-phosphoThr-Val-Ala (K(M) = 3.7 mM) in a reaction that is inhibited competitively by inorganic phosphate (Pi, Ki = 1.6 mM) but unaffected by the product peptide alcohol at concentrations up to 3 mM. The enzyme does not catalyse the incorporation of 18O-label from 18O-labelled water into Pi whether, or not, the product alcohol is present. The dephosphorylated product alcohol of phosphorylated histone. an alternative substrate for the enzyme, serves as a competitive inhibitor for phosphopeptide hydrolysis (Ki = 60 microM) and co-mediates 18O-label exchange into Pi in a concentration-dependent manner (K(M) = 64 microM). These results indicate that hydrolysis occurs through the direct attack of an activated water molecule on the phosphate ester moiety of the substrate in a ternary complex mechanism.
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PMID:Protein phosphatase 1 catalyses the direct hydrolytic cleavage of phosphate monoester in a ternary complex mechanism. 1122 50

Protein phosphatase type 1 (PP1) is a major Ser/Thr protein phosphatase that is involved in many cellular processes. The activity of PP1 is controlled by regulatory subunits, many of which are thought to bind to a hydrophobic groove in PP1 via a short consensus sequence termed the V/IXF motif. To test this hypothesis, 11 variants of yeast PP1 (Glc7) were constructed in which one or more of the residues comprising the groove were changed to alanine. These variants were tested for their biological activity in vivo, for their biochemical activity in vitro, and for their ability to associate with three PP1 binding proteins. Five variants are unable to complement the essential function of PP1 in vivo although they are catalytically active in vitro. Many of the mutants are deficient in binding two V/IXF-containing subunits, Gac1 and Reg1, which regulate glycogen accumulation and glucose repression, respectively, but all retain the ability to associate with Sds22, a regulatory subunit that lacks this motif. The subcellular locations at which PP1 normally accumulates (bud neck, nucleolus, spindle pole body) were not occupied by one PP1 variant. Additionally, we provide evidence that mutations in the hydrophobic groove of PP1 affect substrate specificity. Together, these results demonstrate the importance of the hydrophobic groove for the interaction with regulatory subunits, for the proper subcellular localization of PP1 and for the substrate specificity of PP1.
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PMID:Mutations in yeast protein phosphatase type 1 that affect targeting subunit binding. 1141 94

Total synthesis of the cyclic peptide hepatotoxin motuporin is described, including an efficient synthesis of the constituent amino acid Adda. Three strategies to motuporin are outlined with their relative strengths and weaknesses. Cyclization of the linear peptide precursor was found to proceed moderately well for peptides containing the N-methyldehydrobutyrine residue masked as a threonine, but significant C-terminal epimerization occurred in the presence of the dehydroamino acid. Replacement of the N-methyldehydrobutyrine residue by L-alanine was explored to assess the contribution of this dehydroamino acid to the biochemical activity of motuporin. Some epimerization also was observed during cyclization of the alanine-containing peptide. Synthetic motuporin and both isomers of 5-[L-Ala]-motuporin inhibit the activity of protein phosphatase-1 (PP1) in rat adipocyte lysates with comparable IC(50) values. These results indicate that the N-methyldehydrobutyrine residue is not essential for PP1 inhibition.
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PMID:Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin. 1167 41

The ICP34.5 protein facilitates herpes simplex virus replication by binding and activating protein phosphatase 1 (PP1) by means of a very conserved C-terminal GADD34-like region. Natural variants of the ICP34.5 differing in the number of arginines in an Arg-rich cluster at the N terminus and the number of Pro-Ala-Thr repeats in the central bridge region of the protein were cloned as fusion proteins with a reporter peptide (c-Myc or hrGFP) at the C terminus. The natural variants were obtained from strains differing in passage history, tissue culture behavior, and neuroinvasive disease potential. In transfected cells, these variants localized to different subcellular compartments. The N-terminal Arg-rich cluster acted as a cellular localization signal for discrete regions of the nucleus and cytoplasm, but the ultimate location of ICP34.5 was determined by the number of Pro-Ala-Thr repeats in the central bridge region. PP1 colocalized with the ICP34.5 variant in cells expressing the ICP34.5. The ICP34.5-mediated, herpes simplex virus strain-dependent differences in the modulation of PP1 location and function may be responsible for the strain-associated differences in tissue culture behavior and virulence of the virus.
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PMID:An N-terminal arginine-rich cluster and a proline-alanine-threonine repeat region determine the cellular localization of the herpes simplex virus type 1 ICP34.5 protein and its ligand, protein phosphatase 1. 1178 4

Middle-T antigen is the oncogenic protein of Polyomavirus and associates with several cellular enzymes involved in signal transduction, e.g., Src tyrosine kinases, phosphatidylinositol 3-kinase (PI 3-kinase), protein phosphatase 2A (PP2A), and Shc, an SH2 domain-containing adapter protein. We have shown earlier that middle-T is a target of a cell cycle-regulated serine/threonine-specific kinase, presumably p34cdc2. Phosphorylation of middle-T by p34cdc2 results in increased apparent M, weight of the protein on SDS-polyacrylamide gels. Two threonine residues in positions 160 and 291, respectively, were identified in the middle-T sequence as putative targets of a cyclin-dependent kinase. Replacement of threonine 160 by alanine resulted in a transformation-defective mutant protein that was still capable of forming all the complexes with cellular proteins, suggesting that additional characteristics of middle-T are required for cell transformation. In the present study we report that the defect of the T160A middle-T mutant is compensated by mutations introduced into a domain encompassing amino acids 253 to 302. In particular, mutating serine 283, a canonical phosphorylation site for a cyclin-dependent kinase, to an alanine residue rendered the T160A middle-T mutant wild type. Based on these results we suggest that cell cycle-specific phosphorylation of specific serine and threonine residues by cyclin-dependent kinases regulates middle-T function.
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PMID:Domains in middle-T antigen that cooperate in polyomavirus-mediated oncogenic transformation. 1183 8

The nuclear accumulation of active M-phase promoting factor (MPF) during prophase is thought to be essential for coordinating M-phase events in vertebrate cells. The protein phosphatase Cdc25C, an activator of MPF, enters the nucleus to keep MPF active in the nucleus during prophase. However, the molecular mechanisms that control nuclear translocation of Cdc25C during prophase are unknown. We show that phosphorylation of a serine residue (Ser198) in a nuclear export signal sequence of human Cdc25C occurs during prophase and promotes nuclear localization of Cdc25C. We also show that Polo-like kinase 1 (Plk1) is responsible for this phosphorylation and that constitutively active Plk1 promotes nuclear localization of Cdc25C. Remarkably, a mutant Cdc25C in which Ser198 is replaced by alanine remains in the cytoplasm when wild-type Cdc25C accumulates in the nucleus during prophase. These results suggest that Plk1 phosphorylates Cdc25C on Ser198 and regulates nuclear translocation of Cdc25C during prophase.
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PMID:Plk1 promotes nuclear translocation of human Cdc25C during prophase. 1189 63

Phosphatase IIb (calcineurin, CaN) can reduce N-methyl-D-aspartate (NMDA) synaptic responses by enhancing glycine-independent desensitization. We examined the action of CaN on desensitization in recombinant NMDA receptors comprised of NMDA receptor 1 (NR1) and NR2A subunits. The C-terminus of NR2A, but not NR1, was critical for modulation of desensitization by CaN. Alanine-scanning mutagenesis indicated that serines 900 and 929 in NR2A altered desensitization, as did inhibition of tyrosine phosphatases. Our data suggest that dephosphorylation-dependent regulation of the C-terminus of NR2A increases desensitization of NMDA receptors, providing an additional mechanism for modulation of synaptic signals.
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PMID:Calcineurin acts via the C-terminus of NR2A to modulate desensitization of NMDA receptors. 1198 16


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