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 bcl-2 protein plays an essential role in preventing cell death. Its activity is regulated through association with bcl-2 homologous and nonhomologous proteins and also by serine phosphorylation. We now report that bcl-2 can be proteolytically cleaved towards its N-terminus by a cysteine proteinase present in RL-7 lymphoma cell lysates, yielding a major product of apparent MW 20 kDa, different from the products of bcl-2 cleavage by HIV protease. Moreover, bcl-2 proteins mutated for Asp residues at positions 31 and 34 were efficiently cleaved by RL-7 cell lysates, indicating that this proteolytic activity is distinct from the caspase-3 that cleaves bcl-2 at Asp 34. This bcl-2 cleaving activity is inhibited by E-64 and is therefore distinct from the proteinases of the ICE/Ced-3 family (caspases), whereas reciprocally, ICE (caspase-1) is unable to cleave bcl-2. It is optimally active at pH 5, a feature distinguishing it from calpain, another non-ICE cysteine proteinase which has been associated with apoptosis. This novel bcl-2 cleaving protease, although constitutively present in RL-7 cells and resting peripheral blood lymphocytes (PBL) was upregulated following induction of apoptosis in RL-7 cells or mitogen activation in PBL. The N-terminus of bcl-2 which contains the BH4 domain that binds the kinase Raf-1 and the phosphatase calcineurin is essential for anti-apoptotic activity. Its cleavage might provide a novel post-translational mechanism for regulating bcl-2 function and could amplify ongoing programmed cell death.
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PMID:N-terminus cleavage of bcl-2 by a novel cellular non-ICE cysteine proteinase. 973 98

Inhibitor-1 (I-1), a cyclic AMP-regulated phosphoprotein, inhibits protein phosphatase-1 (PP1) activity in response to hormones. The molecular mechanism for PP1 inhibition by I-1 remains unknown. Mutation of nine acidic residues lining a proposed I-1-binding channel in rabbit PP1alpha yielded one mutant (E256A) slightly impaired in its inhibition by I-1, with the IC50 increased by 3-fold, and one mutant (E275R) located in the beta12-beta13 loop that showed 4-fold enhanced inhibition by I-1. Substituting Tyr-272, a proposed binding site for the toxins okadaic acid and microcystin-LR, in the beta12-beta13 loop with Trp, Phe, Asp, Arg, or Ala impaired PP1alpha inhibition by I-1 by 8-10-fold. Chemical mutagenesis of the Saccharomyces cerevisiae PP1 gene (GLC7) yielded 20 point mutations in the PP1 coding region. Two-hybrid analyses and biochemical assays of these yeast enzymes identified four additional residues in the beta12-beta13 loop that were required for PP1 binding and inhibition by I-1. Ten-fold higher concentrations of I-1 were required to inhibit these mutants. Finally, deletion of the beta12-beta13 loop from PP1alpha maintained full enzyme activity, but attenuated inhibition by I-1 by >100-fold. These data identified the beta12-beta13 loop in the PP1 catalytic subunit as a domain that mediates binding and enzyme inhibition by I-1.
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PMID:Inhibitor-1 interaction domain that mediates the inhibition of protein phosphatase-1. 976 9

The extracellular receptor stimulated kinase ERK2 (p42(MAPK))-phosphorylated human cAMP-specific phosphodiesterase PDE4D3 at Ser579 and profoundly reduced ( approximately 75%) its activity. These effects could be reversed by the action of protein phosphatase PP1. The inhibitory state of PDE4D3, engendered by ERK2 phosphorylation, was mimicked by the Ser579-->Asp mutant form of PDE4D3. In COS1 cells transfected to express PDE4D3, challenge with epidermal growth factor (EGF) caused the phosphorylation and inhibition of PDE4D3. This effect was blocked by the MEK inhibitor PD98059 and was not apparent using the Ser579-->Ala mutant form of PDE4D3. Challenge of HEK293 and F442A cells with EGF led to the PD98059-ablatable inhibition of endogenous PDE4D3 and PDE4D5 activities. EGF challenge of COS1 cells transfected to express PDE4D3 increased cAMP levels through a process ablated by PD98059. The activity of the Ser579-->Asp mutant form of PDE4D3 was increased by PKA phosphorylation. The transient form of the EGF-induced inhibition of PDE4D3 is thus suggested to be due to feedback regulation by PKA causing the ablation of the ERK2-induced inhibition of PDE4D3. We identify a novel means of cross-talk between the cAMP and ERK signalling pathways whereby cell stimuli that lead to ERK2 activation may modulate cAMP signalling.
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PMID:The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579. 1002 32

Even among young, healthy individuals, there is more than a 10-fold variation in insulin sensitivity; however, taken in combination, all the known modifiers of insulin sensitivity - including obesity and a variety of environmental factors - explain less than one third of this variation. It is possible that genetic factors could account for the bulk of the variance observed, and hence play a major role in the development of impaired insulin sensitivity, ie insulin resistance. From the genetic point of view, insulin resistance is thought to be due to the inheritance of a number of mutations in a variety of genes. Three complementary approaches have been applied in the search for mutations: mutational analysis of candidate genes; linkage analysis of candidate genes or chromosomal regions for insulin resistance in familial type 2 diabetes; and random genome mapping with quantitative trait loci (QTL) analysis. Mutational analysis of the insulin signalling cascade has identified a glycine-arginine (Gly-Arg) substitution at codon 972 of the insulin receptor substrate-1 (IRS-1) gene with a carrier prevalence of 9% among Caucasians. Expression of this variant in 32-D cells is associated with a significant (20-30%) impairment of insulin-stimulated PI3-kinase activity, as well as reduced binding of IRS-1 to the p85 regulatory subunit of PI3-kinase. Genotype/phenotype studies stratified according to body mass index (BMI) indicate that obese subjects who are heterozygous for the mutant allele have a 50% decrease in insulin sensitivity, compared with wild-type obese subjects. This suggests that there may be an interaction between the mutant allele and obesity, such that, in the presence of obesity, the mutant variant may aggravate the obesity-associated insulin resistance. Mutational analysis has also shown that homozygous carriers of a codon Met 326 Ile mutation in the p85 subunit of phosphatidylinositol-3 (PI3)-kinase (about 2% of the Caucasian population) have lower glucose tolerance, glucose effectiveness. A further Asp to Tyr polymorphism has been identified at codon 905 of the gene encoding the regulatory subunit of glycogen-associated protein phosphatase-1 (PP1G). Individuals who are heterozygous for this polymorphism constitute 18% of the Caucasian population and appear to exhibit both tissue-specific and pathway-specific insulin resistance. It is likely that inherited insulin resistance will eventually prove to be related to subtle mutations in many such genes of the insulin signalling network and the numerous genetic components controlling energy metabolism.
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PMID:Genetics of insulin resistance. 1032 50

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 - 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 microM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 - 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 - 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 microM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 microM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 - 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.
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PMID:Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid. 1045 72

Site-directed mutagenesis was used to investigate the mechanism of interaction between the catalytic subunit of human protein phosphatase-1 (PP-1cgamma) and members of the calyculin family of toxins. Clavosines A and B are related to calyculins but are glycosylated with a trimethoxy rhamnose group. We provide experimental evidence implicating Tyr-134 as an important residue in PP-1cgamma that mediates interactions with the calyculins. Mutation of Tyr-134 to Phe, to prevent hydrogen bond formation, resulted in a slight increase in sensitivity of PP-1cgamma to clavosines A and B and calyculin A. In contrast, a Y134A mutant was 10-fold less sensitive to inhibition by all three inhibitors. The greatest effect on inhibition was found by substituting an Asp for Tyr-134 in the phosphatase. Clavosine B inhibited PP-1cgamma Y134D with a 310-fold decrease in potency. Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant. These results suggest that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase. The clavosines and calyculin A were tested for their ability to inhibit other mutants of PP-1cgamma (including Ile-133, Val-223, and Cys-291). Our mutagenesis studies provide an experimental basis for assessing models of calyculin binding found in the literature (Lindvall, M. K., Pihko, P. M., and Koskinen, A. M. (1997) J. Biol. Chem. 272, 23312-23316; Gupta, V., Ogawa, A. K., Du, X., Houk, K. N., and Armstrong, R. W. (1997) J. Med. Chem. 40, 3199-3206; Gauss, C. M., Sheppeck, I. J., Nairn, A. C., and Chamberlain, R. (1997) Bioorg. Med. Chem. 5, 1751-1773). A new model for clavosine and calyculin A binding to PP-1c is presented that is consistent with previous structure-function experiments and which accommodates key structural features of the clavosines, including the novel rhamnose moiety.
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PMID:Inhibition of protein phosphatase-1 by clavosines A and B. Novel members of the calyculin family of toxins. 1066 May 82

HePTP is a tyrosine specific protein phosphatase that is strongly expressed in activated T-cells. It was recently demonstrated that in transfected T-cells HePTP impairs TCR-mediated activation of the MAP-kinase family members ERK2 and p38 and it was suggested that both ERK and p38 MAP-kinases are substrates of HePTP. The HePTP gene has been mapped to human chromosome 1q32.1. Abnormalities in this region are frequently found in various hematopoietic malignancies. HePTP is highly expressed in acute myeloid leukemia and its expression in fibroblasts resulted in transformation. To address a possible involvement of HePTP in hematopoietic malignancies we sought to identify HePTP substrate(s) in leukemic cells. Using substrate trapping mutants we have identified the MAP-kinase ERK2 as a specific target of HePTP in the myelogenous leukemia cell line K562. Tyrosine phosphorylated ERK2, but not ERK1, p38, or JNK1, efficiently bound to catalytically inactive HePTP mutants in which the active site cysteine (HePTP-C/S) or the conserved aspartic acid residue (HePTP-D/A) had been exchanged for serine and alanine, respectively. Moreover, the interaction of ERK2 with HePTP trapping mutants was dependent on ERK2 tyrosine phosphorylation, indicating that HePTP is specifically targeted to activated ERK2. Using a deletion mutant of HePTP (HePTP-dLD), in which 14 amino acid residues within the N-terminus are missing, we show that regions outside the catalytic domain are also required for the interaction. Furthermore, overexpression of HePTP in K562 cells and fibroblasts interfered with PMA or growth factor induced MAP-kinase activation and HePTP efficiently dephosphorylated active ERK2 on the tyrosine residue in the activation loop in vitro. Together, these data identify ERK2 as a specific and direct target of HePTP and are consistent with a model in which HePTP negatively regulates ERK2 activity as part of a feedback mechanism. Oncogene (2000) 19, 858 - 869.
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PMID:The MAP-kinase ERK2 is a specific substrate of the protein tyrosine phosphatase HePTP. 1070 94

We examined the possibility that p38 mitogen-activated protein kinase and caspase-3 would be activated for execution of apoptosis and excitotoxicity, the two major types of neuronal death underlying hypoxicischemic and neurodegenerative diseases. Mouse cortical cell cultures underwent widespread neuronal apoptosis 24 h following exposure to 10-30 nM calyculin A, a selective inhibitor of Ser/Thr phosphatase I and IIA. Activity of p38 was increased 2-4 h following exposure to 30 nM calyculin A. Addition of 3-10 microM PD169316, a selective p38 inhibitor, partially attenuated calyculin A neurotoxicity. Activity of caspase-3-like proteases was increased in cortical cell cultures exposed to 30 nM calyculin A for 8-16 h as shown by cleavage of DEVD-p-nitroanilide and phosphorylated tau. Proteolysis of tau was completely blocked by addition of 100 microM N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), a broad-spectrum inhibitor of caspases, but incompletely by 10 microM PD169316. Calyculin A neurotoxicity was partially sensitive to 100 microM z-VAD-fmk. Cotreatment with 10 microM PD169316 and 100 microM z-VAD-fmk showed additive neuroprotection against calyculin A. Neither PD169316 nor z-VAD-fmk showed a beneficial effect against excitotoxic neuronal necrosis induced by exposure to 20 microM NMDA. Thus, caspase-3-like proteases and p38 likely contribute to calyculin A-induced neuronal apoptosis but not NMDA-induced neuronal necrosis.
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PMID:Synergetic activation of p38 mitogen-activated protein kinase and caspase-3-like proteases for execution of calyculin A-induced apoptosis but not N-methyl-d-aspartate-induced necrosis in mouse cortical neurons. 1082 Feb 6

Using an in vitro co-culture assay we found that a rat medullary thymic epithelial cell (TEC) line (TE-R2.5) induces apoptosis of the BWRT8 thymocyte hybridoma (TH) (CD4(hi)CD8(low) alphabetaTCR(hi)). TH apoptosis induced by this TEC line was predominantly mediated by direct cell-cell contacts and was potentiated by cross-linking of the T cell receptor (TCR) by R73 monoclonal antibody (mAb). Dexamethasone (Dx) also triggered TH apoptosis but inhibited death of these cells induced by TE-R2.5 cells or immobilized R73 mAb. The TEC-induced apoptosis was independent of the LFA-1/ICAM-1 interaction but partly depended on a novel 29 kDa molecule expressed on TE-R2.5 cells. All three types of TH apoptosis were followed by the cleavage of poly-(ADP-ribose)-polymerase and were blocked by a caspase inhibitor Z-Val-Ala-Asp(OMe)-CH(2)F.PKC stimulation by phorbol myristate acetate interfered with the TH apoptosis induced by TE-R2.5 and Dx, but did not modulate the effect of R73 mAb. On the contrary, inhibition of calcineurin with cyclosporine A did not influence the apoptosis induced by TE-R2.5 and Dx, but completely prevented the R73-triggered TH cell death. The TE-R2.5-mediated BWRT8 apoptosis was suppressed by Na-orthovanadate, an inhibitor of protein tyrosine phosphatases (PTP) as well as by genistein, a protein tyrosine kinase (PTK) inhibitor, while both compounds potentiated the effect of Dx. Blocking PTP, but not PTK decreased the proapoptotic effect of R73 mAb. These results, including those using a BWRT8 subclone (BWRT8-MDP.2) which is resistant to TCR-triggered apoptosis, but sensitive to apoptosis stimulated by TE-R2.5 and Dx, indicate that TE-R2.5-induced TH apoptosis in our model is different from apoptosis in other TEC co-culture models, published so far.
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PMID:Comparison of signaling pathways involved in apoptosis of a thymocyte hybridoma triggered by a rat thymic medullary epithelial cell line, dexamethasone or T-cell receptor cross-linking. 1084 42

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


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