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

---

Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sequence analysis of the genomes of the Leporipoxviruses myxoma virus and Shope fibroma virus (SFV) led to the discovery of open reading frames homologous to the vaccinia H1L gene encoding a soluble protein phosphatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma BamHI-I fragment and the SFV BamHI-M fragment, and the resulting encoded proteins were designated I1L and M1L, respectively. The localization and orientation of the myxoma I1L and SFV M1L open reading frames within the well conserved central core of the viral genomes closely mirror that of the Orthopoxviruses vaccinia virus and variola virus. The myxoma I1L and SFV M1L phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cysteine, found previously to be essential for phosphotyrosine dephosphorylation. The vaccinia H1L phosphatase was originally shown to have the ability to dephosphorylate phosphotyrosyl and phosphoseryl residues in vitro. To assess whether this is a common feature of poxvirus phosphatases, myxoma I1L was expressed as a GST-fusion protein, purified, and shown to dephosphorylate substrates containing tyrosine and serine phosphorylated residues, in a similar fashion to vaccinia H1L. A myxoma I1L variant, in which the active site cysteine 110 was mutated to serine, was expressed in a parallel fashion to the wild-type I1L protein and found to be completely deficient in its ability to dephosphorylate both phosphotyrosine and phosphoserine amino acids. In an attempt to ascertain the biological requirement for the myxoma I1L phosphatase, we constructed a recombinant myxoma virus containing a disrupted I1L open reading frame. This I1L mutant virus was able to successfully propagate in tissue culture only in the presence of a wild-type complementing gene, and pure virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma I1L dual specificity phosphatase is an essential factor for virus viability.
...
PMID:Myxoma virus and Shope fibroma virus encode dual-specificity tyrosine/serine phosphatases which are essential for virus viability. 783 13

The calcium-dependent interaction between calmodulin (CaM) and the synthetic oligopeptide of a predicted CaM-binding region of human calcineurin A-2 was analysed with an automated surface plasmon resonance biosensor, BIAcore. The oligopeptide was immobilized to a biosensor chip via the amino-terminal cysteine residue by a thiol-disulphide exchange method. The biosensor chip was regenerated by an EGTA-containing buffer after each analysis. Kinetics experiments showed that CaM bound with a high affinity to the oligopeptide in a Ca(2+)-dependent manner. The estimated rate constants of association (kass) and dissociation (kdiss) were 2.3 x 10(5) M-1.s-1 and 3.9 x 10(-3)s-1, respectively. The ratio of kdiss/kass, 1.7 x 10(-8) M, was in good agreement with the dissociation constant (Kd) of 2.4 x 10(-8) M determined from the equilibrium phase.
...
PMID:Real-time-analysis of the calcium-dependent interaction between calmodulin and a synthetic oligopeptide of calcineurin by a surface plasmon resonance biosensor. 792 82

The low M(r) phosphotyrosine protein phosphatase (PTPase) and Yersinia enterocolitica PTPase are inactivated by nitric oxide-generating compounds. Inorganic phosphate, a competitive inhibitor, protects the enzymes from inactivation, suggesting that the action of NO is directed to the active sites. Low M(r) PTPase from bovine liver lost two out of eight thiol groups present in the molecule during the inactivation with sodium nitroprusside and with other NO-producing compounds. The mass spectrometric analyses of tryptic fragments of the enzyme, performed after chemical modification of the NO-unreacted thiol groups, demonstrated that NO caused the oxidation of Cys-12 and Cys-17 to form an S-S bond. A similar reaction was described previously for the reaction of NO with N-methyl-D-aspartate receptor. The NO-inactivated low M(r) PTPase was reactivated by treating the inactive enzyme with thiol-containing reagents. Since all members of the PTPase family have the same reaction mechanism and possess a conserved active site motif that contains an essential cysteine residue, the findings on low M(r) and Yersinia PTPases are potentially interesting for all PTPases, an enzyme class that is involved in a number of important biological processes.
...
PMID:Nitric oxide causes inactivation of the low molecular weight phosphotyrosine protein phosphatase. 792 68

We used the interaction trap, a yeast genetic selection for interacting proteins, to isolate human cyclin-dependent kinase interactor 1 (Cdi1). In yeast, Cdi1 interacts with cyclin-dependent kinases, including human Cdc2, Cdk2, and Cdk3, but not with Ckd4. In HeLa cells, Cdi1 is expressed at the G1 to S transition, and the protein forms stable complexes with Cdk2. Cdi1 bears weak sequence similarity to known tyrosine and dual specificity phosphatases. In vitro, Cdi1 removes phosphate from tyrosine residues in model substrates, but a mutant protein that bears a lesion in the putative active site cysteine does not. Overexpression of wild-type Cdi1 delays progression through the cell cycle in yeast and HeLa cells; delay is dependent on Cdi1 phosphatase activity. These experiments identify Cdi1 as a novel type of protein phosphatase that forms complexes with cyclin-dependent kinases.
...
PMID:Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. 824 50

Phosphoseryl/threonyl protein phosphatase inhibitors, viz. okadaic acid and calyculin-A, failed to induce nuclear factor-kappa B (NF-kappa B) nuclear translocation in several primary human cells although a marked and rapid induction was observed in their simian virus 40 transformed counterparts. Inability to induce NF-kappa B cannot be due to a non-activatable system since NF-kappa B was strongly activated by tumor necrosis factor in all the five primary cell types tested. It is also unlikely that the differential induction was due to differential sensitivity of primary and transformed cells to phosphatase inhibitors as the intracellular phosphatase activities of both cell types were equally inhibited by these inhibitors. However, pretreatment with hydrogen peroxide or buthionine sulfoximine, chemicals known to directly or indirectly elevate the intracellular free-radical levels, enabled okadaic acid to induce nuclear translocation of NF-kappa B in primary cells. Conversely cysteine, an antioxidant and precursor of the free radical scavenger, glutathione, inhibited the induction of NF-kappa B by tumor necrosis factor in primary cells, and by okadaic acid or tumor necrosis factor in transformed cells. These data, taken together, suggest that free radical-dependent oxidation and protein phosphorylation are not independent modes of NF-kappa B induction, but are both required for the release of NF-kappa B from I kappa B. Furthermore, the differential induction of NF-kappa B nuclear translocation by okadaic acid in primary and transformed human cells, reported herein, reflects intrinsic differences in the intracellular oxidative state between the two groups of cells. The induction of NF-kappa B by tumor necrosis factor in primary cells suggests that this cytokine fulfills the requirement for oxidation, possibly by inducing the production of free radicals.
...
PMID:Differential induction of nuclear NF-kappa B by protein phosphatase inhibitors in primary and transformed human cells. Requirement for both oxidation and phosphorylation in nuclear translocation. 825 18

Chemical modification of the sulfhydryl residues of the catalytic subunits of protein phosphatases 1 and 2A was studied. Both enzymes were inactivated by a variety of thiol group reagents. Mercurial compounds were the most effective inactivators. Of the alkylating agents the maleimides were more effective than iodoacetate or iodoacetamide which were relatively slow reacting. Both enzymes were also inactivated by disulfides, including glutathione disulfide, 5,5'-dithiobis(2-nitrobenzoic acid), and 4,4'-dipyridyl disulfide. The latter two were much more reactive than glutathione disulfide and, in addition, displayed a significant differential reactivity toward phosphatase 1. The apparent second-order rate constants for the inactivation of phosphatase 1 were 20- to 70-fold higher than for phosphatase 2A with 5,5'-dithiobis(2-nitrobenzoic acid) and 4,4'-dipyridyl disulfide. Kinetic analysis indicated that inactivation of both enzymes could be correlated with the modification of one cysteine per one mole of enzyme.
...
PMID:Reactivity of sulfhydryl groups of the catalytic subunits of rabbit skeletal muscle protein phosphatases 1 and 2A. 838 Sep 64

We have reacted calmodulins containing cysteines substituted at positions 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate intramolecularly cross-linked proteins termed BMHCM or BMHCM1, respectively. Reactions were also performed with N-ethylmaleimide (NEM) in place of BMH to generate corresponding S-ethylsuccinimidylated proteins termed NEMCM or NEMCM1. The abilities of these proteins to activate plant NAD kinase, erythrocyte Ca(2+)-ATPase and bovine brain calcineurin activities were assessed. The BMH- or NEM-reacted proteins activate calcineurin activity as does control calmodulin. Kact values for Ca(2+)-ATPase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in Vmax values. Activation of this enzyme by NEMCM or NEMCM1 is not different from the control. In NAD kinase activation experiments BMHCM and BMHCM1 are associated with a 10 to 20-fold increase in Kact values and a 60-75% reduction in Vmax values relative to the control. NEMCM1 is not associated with any apparent changes in NAD kinase activation, however, NEMCM is associated with a 10-fold increase in the Kact value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased Kact value, implying that this change is due to interactions between S-(ethylsuccinimido)cysteines at positions 3 and 146. In conclusion, cross-linking and associated distortions in the structure of calmodulin appear to have little or no effect on activation of calcineurin enzyme activity. However, bending in the central helix and/or steric restrictions associated with cross-linking increase significantly the Kact value for Ca(2+)-ATPase and NAD kinase activation, and dramatically reduce maximal activation of NAD kinase activity.
...
PMID:Activation of enzymes by calmodulins containing intramolecular cross-links. 838 93

The expression of the human CL100 gene and its mouse homologue 3CH134 is increased up to 40-fold in fibroblasts exposed to oxidative/heat stress and growth factors. CL100 is a member of an expanding family of protein tyrosine phosphatases with amino acid sequence similarity to a Tyr/Ser-protein phosphatase encoded by the late H1 gene of vaccinia virus. Here we show that the CL100 phosphatase, expressed and purified in bacteria, rapidly and potently inactivates recombinant MAP kinase in vitro by the concomitant dephosphorylation of both its phosphothreonine and phosphotyrosine residues. Furthermore, CL100 suppresses the [val12] ras-induced activation of MAP kinase in a cell-free system from Xenopus oocytes. Both activities are abolished by mutagenesis of the highly conserved cysteine (Cys-258) within the phosphatase active site. In contrast to the vaccinia H1 phosphatase, CL100 shows no measurable catalytic activity towards a number of other substrate proteins modified on serine, threonine or tyrosine residues. Our results demonstrate that CL100 is a dual specificity phosphatase and indicate that MAP kinase is one of its physiological targets. CL100 may be the first example of a new class of protein phosphatases responsible for modulating the activation of MAP kinase following exposure of quiescent cells to growth factors and further implicates MAP kinase activation/deactivation in the cellular response to stress.
...
PMID:The human CL100 gene encodes a Tyr/Thr-protein phosphatase which potently and specifically inactivates MAP kinase and suppresses its activation by oncogenic ras in Xenopus oocyte extracts. 839 41

Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the first Ca(2+) ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype. In the present work we have studied the functional and structural consequences of the Phe-92 --> Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM. Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca(2+) at the C-terminal domain. To investigate the structural consequences of the F92A mutation, we constructed four recombinant C-terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca(2+)-free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92. The F92A mutation causes a decrease in stability of the domain in the absence of Ca(2+) as indicated by an 11.8 degree C shift in the far UV circular dichroism thermal unfolding curve. This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca(2+)-dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid. The fluorescence increase induced by C-CaMF92A is approximately 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca(2+). The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca(2+) affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca(2+) binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an important contribution to the Ca(2+)-induced transition in the C-terminal domain of CaM. This is most likely the reason for the severely impaired regulatory properties of the CaM mutants having Ala substituted for Phe-92.
...
PMID:The role of Phe-92 in the Ca(2+)-induced conformational transition in the C-terminal domain of calmodulin. 862 80

Calcium-dependent regulation of intracellular processes is mediated by proteins that on binding Ca2+ assume a new conformation, which enables them to bind to their specific target proteins and to modulate their function. Calmodulin (CaM) and troponin C, the two best characterized Ca2+-regulatory proteins, are members of the family of Ca2+-binding proteins utilizing the helix-loop-helix structural motif (EF-hand). Herzberg, Moult, and James (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) proposed that the Ca2+-induced conformational transition in troponin C involves opening of the interface between the alpha-helical segments in the N-terminal domain of this protein. Here we have tested the hypothesis that a similar transition is the key Ca2+-induced regulatory event in calmodulin. Using site-directed mutagenesis we have substituted cysteine residues for Gln41 and Lys75 (CaM41/75) or Ile85 and Leu112 (CaM85/112) in the N-terminal and C-terminal domains, respectively, of human liver calmodulin. Based on molecular modeling, cysteines at these positions were expected to form intramolecular disulfide bonds in the Ca2+-free conformation of the protein, thus blocking the putative Ca2+-induced transition. We found that intramolecular disulfide bonds are readily formed in both mutants causing a decrease in affinity for Ca2+ and the loss of ability to activate target enzymes, phosphodiesterase and calcineurin. The regulatory activity is fully recovered in CaM41/75 and partially recovered in CaM85/112 upon reduction of the disulfide bonds with dithiothreitol and blocking the Cys residues by carboxyamidomethylation or cyanylation. These results indicate that the Ca2+-induced opening of the interfaces between helical segments in both domains of CaM is critical for its regulatory properties consistent with the Herzberg-Moult-James model.
...
PMID:Blocking the Ca2+-induced conformational transitions in calmodulin with disulfide bonds. 863 77


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---