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)

Phosphorylation and dephosphorylation events may critically control junction assembly and stability, as well as regulate the formation of the cadherin-cytoskeleton complex, thus influencing the adhesive function of cells. In the present study, we have used specific activators and inhibitors of protein kinases and phosphatases to analyze the role of protein phosphorylation in the maintenance of epithelial architecture. Okadaic acid and calyculin A cell treatments induced two major effects: a dramatic alteration of the keratin network of epidermal cells and a complete disruption of cell-cell contacts. This loss in cell-cell contacts was not tissue and species restricted and the interactions of keratinocytes with the matrix were not involved. The observed changes were highly specific for these drugs and were obtained in the range of concentrations corresponding to the inhibition of protein phosphatase 1 (PP1). They were time- and dose-dependent, and reversible, excluding a cytotoxic effect of the drugs. A decrease in electrophoretic mobility of beta-catenin, a major protein involved in the regulation of intercellular adherens junctions, was observed in keratinocytes and fibroblasts treated with okadaic acid and calyculin A, suggesting a change in the protein phosphorylation level and/or protein conformation. Data from beta-catenin immunocomplex autoradiography performed after 32P in vivo incorporation in untreated and okadaic acid or calyculin A-treated HaCaT cells, demonstrated a higher level of phosphorylation of beta-catenin in treated cells compared to untreated ones. Analysis of 32P-labeled phosphoaminoacids demonstrated that beta-catenin was exclusively phosphorylated on serine-threonine residues but not on tyrosine residues. Immunoprecipitations and Western blotting using anti-phosphoserine and anti-phosphotyrosine antibodies confirmed these data. The change in beta-catenin phosphorylation on serine-threonine residues may play a role in the control of the cohesion between epithelial cells and may be involved in the regulation of the transduction signal.
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PMID:Hyperphosphorylation of beta-catenin on serine-threonine residues and loss of cell-cell contacts induced by calyculin A and okadaic acid in human epidermal cells. 905 23

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacted with APC in the yeast two-hybrid system. Expression of B56 reduced the abundance of beta-catenin and inhibited transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin was blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling.
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PMID:Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. 1009 33

Axin forms a complex with glycogen synthase kinase-3beta (GSK-3beta) and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin, thereby stimulating the degradation of beta-catenin. Because GSK-3beta also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3beta inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3beta were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3beta, inhibited the phosphorylation of Axin by GSK-3beta in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated beta-catenin in L cells and expression of Dvl-1(DeltaPDZ), in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl.
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PMID:Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability. 1019 36

The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are necessary for Wnt signal transduction. Utilizing the yeast two-hybrid system, we identified protein phosphatase 2Calpha (PP2C) as a Dvl-PDZ domain-interacting protein. PP2C exists in a complex with Dvl, beta-catenin, and Axin, a negative regulator of Wnt signaling. In a Wnt-responsive LEF-1 reporter gene assay, expression of PP2C activates transcription and also elicits a synergistic response with beta-catenin and Wnt-1. In addition, PP2C expression relieves Axin-mediated repression of LEF-1-dependent transcription. PP2C utilizes Axin as a substrate both in vitro and in vivo and decreases its half-life. These results indicate that PP2C is a positive regulator of Wnt signal transduction and mediates its effects through the dephosphorylation of Axin.
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PMID:Protein phosphatase 2Calpha dephosphorylates axin and activates LEF-1-dependent transcription. 1064 91

Axin forms a complex with adenomatous polyposis coli gene product (APC), glycogen synthase kinase-3beta (GSK-3beta), and beta-catenin through different binding sites and downregulates beta-catenin. GSK-3beta-dependent phosphorylation of APC-(1211-2075) which has the Axin-binding site was facilitated by Axin, but that of APC-(959-1338) which lacks the Axin-binding site was not. Axin-(298-506) or Axin-(298-832), which has the GSK-3beta- and beta-catenin- but not APC-binding sites, did not enhance GSK-3beta-dependent phosphorylation of either APC-(1211-2075) or APC-(959-1338). Furthermore, beta-catenin stimulated the phosphorylation of APC-(959-1338) and APC-(1211-2075) by GSK-3beta in the presence of Axin. Consistent with these in vitro observations, expression of beta-catenin or Axin in COS cells promoted an SDS gel band shift of APC. These results indicate that APC complexed with Axin is effectively phosphorylated by GSK-3beta and that beta-catenin may modulate this phosphorylation. In addition, the heterodimeric form of protein phosphatase 2A (PP2A) directly bound to Axin, and PP2A complexed with Axin dephosphorylated APC phosphorylated by GSK-3beta. Taken together, these results suggest that GSK-3beta-dependent phosphorylation of APC can be modulated by beta-catenin and PP2A complexed with Axin.
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PMID:GSK-3beta-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by beta-catenin and protein phosphatase 2A complexed with Axin. 1069 23

Protein phosphatase 2A (PP2A) plays central roles in development, cell growth and transformation. Inactivation of the gene encoding the PP2A catalytic subunit Calpha by gene targeting generates a lethal embryonic phenotype. No mesoderm is formed in Calpha(-/-) embryos. Here, we found that during normal early embryonic development Calpha was predominantly present at the plasma membrane whereas the highly homologous isoform Cbeta was localized to the cytoplasm and nuclei, suggesting the inability of Cbeta to compensate for vital functions of Calpha in Calpha(-/-) embryos. In addition, PP2A was found in a complex containing the PP2A substrates E-cadherin and beta-catenin. In Calpha(-/-) embryos, E-cadherin and beta-catenin were redistributed from the plasma membrane to the cytosol. Cytosolic concentrations of beta-catenin were low. Our results suggest that Calpha is required for stabilization of E-cadherin/beta-catenin complexes at the plasma membrane.
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PMID:Distinct role of protein phosphatase 2A subunit Calpha in the regulation of E-cadherin and beta-catenin during development. 1078 42

Hypoxia activates a number of gene products through degradation of the transcriptional coactivator cAMP response element binding protein (CREB). Other transcriptional regulators (e.g., beta-catenin and NF-kappa B) are controlled through phosphorylation-targeted proteasomal degradation, and thus, we hypothesized a similar degradative pathway for CREB. Differential display analysis of mRNA derived from hypoxic epithelia revealed a specific and time-dependent repression of protein phosphatase 1 (PP1), a serine phosphatase important in CREB dephosphorylation. Subsequent studies identified a previously unappreciated proteasomal-targeting motif within the primary structure of CREB (DSVTDS), which functions as a substrate for PP1. Ambient hypoxia resulted in temporally sequential CREB serine phosphorylation, ubiquitination, and degradation (in vitro and in vivo). HIV-tat peptide-facilitated loading of intact epithelia with phosphopeptides corresponding to this proteasome targeting motif resulted in inhibition of CREB ubiquitination. Further studies revealed that PP1 inhibitors mimicked hypoxia-induced gene expression, whereas proteasome inhibitors reversed the hypoxic phenotype. Thus, hypoxia establishes conditions that target CREB to proteasomal degradation. These studies may provide unique insight into a general mechanism of transcriptional regulation by hypoxia.
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PMID:Phosphorylation-dependent targeting of cAMP response element binding protein to the ubiquitin/proteasome pathway in hypoxia. 1103 95

Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of beta-catenin and cyclin D1), Alzheimer's disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ss, beta-catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419-672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These "Axin-His6" matrices were found to selectively bind recombinant rat GSK-3 beta and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes.
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PMID:Purification of GSK-3 by affinity chromatography on immobilized axin. 1108 79

Wnt signaling increases beta-catenin abundance and transcription of Wnt-responsive genes. Our previous work suggested that the B56 regulatory subunit of protein phosphatase 2A (PP2A) inhibits Wnt signaling. Okadaic acid (a phosphatase inhibitor) increases, while B56 expression reduces, beta-catenin abundance; B56 also reduces transcription of Wnt-responsive genes. Okadaic acid is a tumor promoter, and the structural A subunit of PP2A is mutated in multiple cancers. Taken together, the evidence suggests that PP2A is a tumor suppressor. However, other studies suggest that PP2A activates Wnt signaling. We now show that the B56, A and catalytic C subunits of PP2A each have ventralizing activity in Xenopus embryos. B56 was epistatically positioned downstream of GSK3beta and axin but upstream of beta-catenin, and axin co-immunoprecipitated B56, A and C subunits, suggesting that PP2A:B56 is in the beta-catenin degradation complex. PP2A appears to be essential for beta-catenin degradation, since beta-catenin degradation was reconstituted in phosphatase-depleted Xenopus egg extracts by PP2A, but not PP1. These results support the hypothesis that PP2A:B56 directly inhibits Wnt signaling and plays a role in development and carcinogenesis.
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PMID:Protein phosphatase 2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus. 1148 15

Wnt signaling plays a key role in cell proliferation and development. Recently, casein kinase I (CKI) and protein phosphatase 2A (PP2A) have emerged as positive and negative regulators of the Wnt pathway, respectively. However, it is not clear how these two enzymes with opposing functions regulate Wnt signaling. Here we show that both CKI delta and CKI epsilon interacted directly with Dvl-1, and that CKI phosphorylated multiple components of the Wnt-regulated beta-catenin degradation complex in vitro, including Dvl-1, adenomatous polyposis coli (APC), axin, and beta-catenin. Comparison of peptide maps from in vivo and in vitro phosphorylated beta-catenin and axin suggests that CKI phosphorylates these proteins in vivo as well. CKI abrogated beta-catenin degradation in Xenopus egg extracts. Notably, CKI decreased, whereas inhibition of CKI increased, the association of PP2A with the beta-catenin degradation complex in vitro. Additionally, inhibition of CKI in vivo stabilized the beta-catenin degradation complex, suggesting that CKI actively destabilizes the complex in vivo. The ability of CKI to induce secondary body axes in Xenopus embryos was reduced by the B56 regulatory subunit of PP2A, and kinase-dead CKI epsilon acted synergistically with B56 in inhibiting Wnt signaling. The data suggest that CKI phosphorylates and destabilizes the beta-catenin degradation complex, likely through the dissociation of PP2A, providing a mechanism by which CKI stabilizes beta-catenin and propagates the Wnt signal.
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PMID:Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. 1181 47


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