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)

We studied the effect of protein phosphatase and kinase inhibitors on Tax-mediated transcription of constructs carrying the reporter gene chloramphenicol acetyl transferase under the control of either the full-length LTR of HTLV-I or three copies of the tax-responsive 21-bp repeats. We observed that treatment with okadaic acid, which inhibits the serine/threonine protein phosphatases type 1 and 2A, reduced HTLV-I LTR transcriptional activation in MT2 and K562 cells; on the contrary, the enhancer activity of the 21-bp sequences was significantly increased in both cell lines; treatment with the protein kinase C inhibitor H-7 blocked Tax-mediated transcription of both constructs. We also found that treatment with sodium orthovanadate, a tyrosine phosphatase inhibitor, reduced Tax-mediated activation of both plasmids. These findings indicated that specific serine/threonine phosphorylation events are required for Tax-mediated HTLV-I LTR activation and also suggested that phosphorylation at tyrosine residues is involved in this process.
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PMID:Tax-induced HTLV-I LTR transcriptional activation is modulated by phosphorylation. 799 95

INH, a type 2A protein phosphatase (PP2A), negatively regulates entry into M phase and the cyclin B-dependent activation of cdc2 in Xenopus extracts. INH appears to be central to the mechanism of the trigger for mitotic initiation, as it prevents the premature activation of cdc2. We first show that INH is a conventional form of PP2A with a B alpha regulatory subunit. We next explore the mechanism by which it inhibits cdc2 activation by examining the effect of purified PP2A on the reaction pathways controlling cdc2 activity. Our results suggest that although PP2A inhibits the switch in tyrosine kinase and tyrosine phosphatase activities accompanying mitosis, this switch is a consequence of the inhibition of some other rate-limiting event. In the preactivation phase, PP2A inhibits the pathway leading to T161 phosphorylation, suggesting that this activity may be one of the rate-limiting events for transition. However, our results also suggest that the accumulation of active cdc2/cyclin complexes during the lag is only one of the events required for triggering entry into mitosis.
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PMID:Inhibition of cdc2 activation by INH/PP2A. 804 24

We have examined the negative regulation of the 44-kDa mitogen-activated protein kinase (MAP kinase), also known as extracellular signal-regulated protein kinase 1 (ERK1), in NIH3T3 cells transfected with an expression plasmid encoding the human insulin receptor (NHIR cells). In these cells ERK1 activation is induced by two distinct stimuli, insulin and tumor-promoting agent (TPA). While insulin was found to be more potent than TPA for ERK1 activation, both stimuli produced the same transient activation pattern with a rapid peak (reached within 5 min) followed by a fast decrease within 20 min. By performing reconstitution experiments with immunoprecipitated ERK1 and lysates from NHIR cells, we showed that extracts from untreated cells exhibit an ERK1 inhibitory activity. Interestingly, this inhibitor was found to be regulated by insulin and TPA with a profile that is the mirror image of ERK1 activity. This repressing activity was sensitive to tyrosine phosphatase inhibitors, such as sodium orthovanadate and zinc acetate, but it was not affected by serine/threonine phosphatase inhibitors, such as sodium fluoride and okadaic acid. Moreover, it was possible to observe in extracts of NHIR cells an activity dephosphorylating ERK1. The time course of this phosphatase activity was comparable to that of the ERK1 inhibition, suggesting that the repressing activity could reflect a dephosphorylating action. Interestingly, phosphatase 2A treatment of extracts from 5-min TPA-treated cells (where the ERK1 inhibitor was weak) was able to induce an increase in the ERK1 repressing activity. This suggests that serine/threonine dephosphorylation of ERK1 inhibitor leads to an increase in its activity. In summary, we have shown that NHIR cells contain a regulatable ERK1 inhibitor, which is likely to be due to tyrosine phosphatase(s). We would like to suggest that such activities are key components in the fine-tuning of the MAP kinase cascade.
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PMID:Insulin and tumor-promoting agent regulate an inhibitor of the 44-kDa mitogen-activated protein kinase/extracellular signal-regulated protein kinase 1 in fibroblasts. 828 32

We have characterized a growth factor-inducible gene, erp, and demonstrated that it encodes a 367-amino-acid nontransmembrane tyrosine phosphatase protein with significant similarity to the vaccinia virus H1 protein. Immunoprecipitation analyses show that the erp protein, ERP, is rapidly induced following serum stimulation of quiescent fibroblasts. ERP has been expressed as a fusion protein with glutathione S-transferase and shown to have tyrosine as well as serine protein phosphatase activity. The enzymatic activity of ERP depends on the presence of reducing agents such as dithiothreitol, and its tyrosine phosphatase activity is inhibited by sodium vanadate, a potent inhibitor of protein tyrosine phosphatases. The number of stable NIH 3T3 clones obtained after transfection with a vector expressing the complete ERP protein is reduced more than 90% compared with that after transfection with a vector expressing a mutated inactive ERP protein. The remaining ERP-expressing clones present a significant increase in the proportion of bi- and multinucleated cells and a decrease in proliferation rate. Studies on the genomic structure reveal that the erp transcription unit is 2.8 kbp long and split into four exons. The erp gene maps to the 17A2-17C region of the murine genome. Our results demonstrate that the protein product of the immediate-early gene erp has a negative effect on cell proliferation.
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PMID:Structure, mapping, and expression of erp, a growth factor-inducible gene encoding a nontransmembrane protein tyrosine phosphatase, and effect of ERP on cell growth. 835 78

To understand the role of the type 2A-like protein phosphatase in the cell division cycle, we investigated the mutant phenotypes obtained when the fission yeast ppa1+ and ppa2+ phosphatase genes (which encode polypeptides with approximately 80% identity to mammalian type 2A phosphatases) were either deleted or overexpressed. We also investigated the in vivo effect of okadaic acid, an inhibitor of protein serine/threonine phosphatases, on cell division. We show that ppa2+ interacts genetically with the cell cell regulators cdc25+ and wee1+, as a ppa2 deletion is lethal when combined with wee1-50 but partially suppresses the conditional lethality of cdc25-22 mutation. Evidence that ppa2+ negatively controls the entry into mitosis, possibly through the regulation of cdc2 tyrosine phosphorylation, is presented. ppa2 phosphatase is abundant in the cytoplasm, in contrast to the type 1-like phosphatase dis2, which is enriched in the nucleus. Overproduced ppa1 or ppa2 proteins accumulate in the cytoplasm near the nuclear periphery, and cells arrest in interphase. Okadaic acid-treated cells, like a ppa2 deletion, are short in length and display protein hyperphosphorylation. Cytokinesis is also inhibited, producing binucleated cells. We show that ppa2 is the genetic locus controlling okadaic acid sensitivity. The ppa2 deletion reveals the same hyperphosphorylated proteins as okadaic acid. When a strain deleted for ppa2 is treated with okadaic acid, cell size is reduced further to that of wee1-50 mutant strain or overexpressing the cdc25+ gene product, suggesting functional relationship of ppa2 with the cdc25 tyrosine phosphatase and/or the wee1 kinase in cell cycle control.
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PMID:Negative regulation of mitosis by the fission yeast protein phosphatase ppa2. 838 6

Calcineurin is shown to hydrolyze 2,2,2-trifluoroethyl phosphate. The time course of hydrolysis showed a lag period. The lag was present at all substrate concentrations used and was dependent on substrate concentration. Evaluation of the kinetic constants from the steady state portion of the enzymatic reaction indicated that hydrolysis of this compound fit the Bronsted relationship determined previously for Vmax/Km term for the hydrolysis of aromatic phosphate esters. Moreover, a Bronsted relationship for the Vmax was developed for the substrates containing leaving groups that are more basic than 2,3,4,5-tetrafluorotyrosine. The hydrolysis of 2,3,4,5-tetrafluorotyrosine phosphate did not fit this relationship implying a change in the rate limiting step with this substrate. The significance of these data to the serine and tyrosine phosphatase activities of calcineurin is discussed. A rationale for understanding the determinant of both activities of calcineurin is presented.
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PMID:Hydrolysis of trifluoroethyl phosphate as evidence that the serine and tyrosine phosphatase activities of calcineurin share the same specificity determinant. 839 34

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.
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PMID:The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site. 841

Transforming growth factor beta 1 (TGF-beta 1) exerts a positive effect on the transcription of genes coding for several extracellular matrix-related products, including collagen I. We have previously identified a strong TGF-beta 1-responsive element (TbRE) in the upstream promoter sequence of the alpha 2(I) collagen (COL1A2) gene. Our experiments have shown that TGF-beta 1 stimulates COL1A2 transcription by increasing binding of an Sp1-containing complex (TbRC) to the TbRE. They have also suggested that the change occurs via posttranslational modification of a protein(s) directly or indirectly interacting with Sp1. Here, we provide evidence showing that tyrosine dephosphorylation of nuclear proteins mimics the stimulation of COL1A2 transcription by the TGF-beta 1-activated signaling pathway. Preincubation of nuclear extracts with protein tyrosine phosphatase (PTPase) but not with protein phosphatase type 2A (PP2A), a serine/threonine phosphatase, enhanced binding of the TbRC to the same degree as culturing cells in TGF-beta 1. Consistent with these in vitro findings, genistein, a tyrosine kinase inhibitor, led to markedly increased COL1A2 gene expression, whereas sodium orthovanadate, a tyrosine phosphatase inhibitor, decreased it substantially. These results were supported by transfection experiments showing that genistein and sodium orthovanadate have opposite effects on TbRE-mediated transcription. Moreover, nuclear proteins isolated from genistein-treated cells were found to interact with the TbRE significantly more than those from untreated cells. Furthermore, pretreatment of cells with sodium orthovanadate virtually abrogated nuclear protein binding to the TbRE, but not to a neighboring cis-acting element unresponsive to TGF-beta 1. The results of this study, therefore, provide the first correlation between tyrosine dephosphorylation, increased binding of a transcriptional complex, and TGF-beta 1 stimulation of gene expression.
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PMID:Tyrosine dephosphorylation of nuclear proteins mimics transforming growth factor beta 1 stimulation of alpha 2(I) collagen gene expression. 852 47

The recent discovery of the vaccinia virus protein phosphatase VH1, and its mammalian counterparts has highlighted a novel subfamily of protein tyrosine phosphatases that exhibit dual specificity toward phosphotyrosine- and phosphoserine/threonine-residues. We have identified further members of this subfamily. The characterisation of one clone in particular, which we have named threonine-tyrosine phosphatase 1 (TYP 1), encodes a protein homologous to CL100, but differs dramatically in its regulation. TYP 1 is not expressed in human fibroblasts unlike other CL100-like genes. Furthermore, northern analysis has demonstrated that following mitogenic stimulation of squamous cells, induction of TYP 1 mRNA reaches its maximal levels after four hours, in contrast to the immediate early CL100-like genes. Both TYP 1 and CL100 mRNAs are induced upon TGF-beta treatment of squamous cell lines sensitive to the growth factors antiproliferative effects. When TYP 1 is transfected into COS-1 cells, the gene product inhibits both ERK2 and p54 MAP kinase subfamilies. In addition, we show that purified TYP 1 protein efficiently inactivates recombinant ERK2 in vitro by the concomitant dephosphorylation of both its phosphothreonine and -tyrosine residues. TYP 1 encodes a nuclear protein, which when expressed in COS cells is stabilised by EGF treatment.
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PMID:Isolation and characterisation of a uniquely regulated threonine, tyrosine phosphatase (TYP 1) which inactivates ERK2 and p54jnk. 854 12

Tumor necrosis factor-alpha (TNF-alpha) is a proposed mediator of insulin resistance in obese/diabetic animals through its effects on tyrosine phosphorylation of the insulin receptor and its substrate, insulin receptor substrate-1. In this study, the acute effects of TNF-alpha on the mitogen-activated protein kinase (MAPK) signalling cascade were examined in cultured rat skeletal muscle cell line, L6. Insulin treatment of L6 cells resulted in a rapid increase in MAPK activity (> twofold in 5 min with 10 nM insulin). Prior treatment with TNF-alpha for 60 min blocked subsequent insulin-induced activation of MAPK in a dose- and time-dependent manner. Metabolic labelling studies with inorganic [32P]phosphate followed by immuno-precipitation of MAPK and its upstream activator, mitogen-activated protein kinase kinase, indicated decreased phosphorylation of MAPK and its kinase in response to insulin in cells exposed to TNF-alpha. This effect of TNF-alpha was not due to inhibition of insulin-stimulated p21ras-GTP loading or Raf-1 phosphorylation. Low concentrations (2 nM) of okadaic acid, a serine/threonine phosphatase inhibitor, prevented TNF-alpha-induced inhibition of MAPK and restored insulin's effect on MAPK activity, while orthovanadate (a tyrosine phosphatase inhibitor), inhibitor 2 (phosphatase-1 inhibitor) and FK506 (phosphatase-2B inhibitor) were ineffective. These results suggested an involvement of an okadaic-acid-sensitive serine/threonine phosphatase in TNF-alpha-induced blockade of insulin's effect on MAPK and/or its kinase. Therefore, we examined the effect of TNF-alpha on protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) activities. As reported by us earlier, insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in control cells. TNF-alpha treatment blocked insulin-induced activation of PP-1. In contrast to PP-1, TNF-alpha caused a 60% increase in PP-2A activity and insulin failed to prevent this TNF-alpha effect. The time course of PP-2A activation by TNF-alpha preceded the kinetics of inhibition of MAPK. Cell-permeable ceramide analogs mimicked the TNF-alpha effect on MAPK inhibition and PP-2A activation. We conclude that TNF-alpha abrogates the insulin effect on MAPK activation by increasing dephosphorylation of MAPK kinase via an activated phosphatase.
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PMID:Effect of tumor necrosis factor-alpha on insulin-stimulated mitogen-activated protein kinase cascade in cultured rat skeletal muscle cells. 866 40


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