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)

Stimulation of the T-cell antigen receptor (TCR) induces activation of multiple tyrosine kinases, resulting in phosphorylation of numerous intracellular substrates. One substrate is p95vav, which is expressed exclusively in hematopoietic and trophoblast cells. It contains a number of structural motifs, including Src homology 2, Src homology 3, and pleckstrin homology domains and a putative guanine nucleotide exchange domain. The role of p95vav in TCR-mediated signaling processes is unclear. Here, we show that overexpression of p95vav alone in Jurkat T cells leads to activation of the nuclear factors, including NFAT, involved in interleukin-2 expression. Furthermore, p95vav synergizes with TCR stimulation in inducing NFAT- and interleukin-2-dependent transcription. In contrast, NFAT activation by a G-protein-coupled receptor is not modulated by p95vav overexpression, suggesting that the effect is specific to the TCR signaling pathways. Although removal of the first 67 amino acids of p95vav activates its transforming potential in NIH 3T3 cells, this region appears to be required for its function in T cells. We further demonstrate that the p95vav-induced NFAT activation is not mimicked by Ras activation, though its function is dependent upon Ras and Raf. Furthermore, the activating function of p95vav is blocked by FK506, suggesting that its activity also depends on calcineurin. To further dissect p95vav involvement in TCR signaling, we analyzed various Jurkat mutants deficient in TCR signaling function or TCR expression and showed that an intact TCR signaling pathway is required for p95vav to function. However, overexpression of p95vav does not appear to influence TCR-induced protein tyrosine phosphorylation or increases in cytoplasmic free calcium. Taken together, our data suggest that p95vav plays an important role at an yet unidentified proximal position in the TCR signaling cascade.
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PMID:A functional T-cell receptor signaling pathway is required for p95vav activity. 762 28

Pleckstrin is the major substrate phosphorylated on serine and threonine in response to stimulation of human platelets by thrombin (Abrams, C. S., Zhao, W., Belmonte, E., and Brass, L. F. (1995) J. Biol. Chem. 270, 23317-23321). We now show that pleckstrin in platelets is in a complex with inositol polyphosphate 5-phosphatase I (5-phosphatase I). This enzyme hydrolyzes the 5-phosphate from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thus serves as a calcium signal-terminating enzyme, since the substrates but not the products mobilize intracellular calcium. Pleckstrin co-immunoprecipitates with 5-phosphatase I in homogenates of platelets. Platelet homogenates fractionated by anion exchange chromatography show co-elution of pleckstrin and 5-phosphatase I. Fractions containing phosphorylated pleckstrin have 7-fold greater 5-phosphatase activity than those containing unphosphorylated pleckstrin. Mixing experiments with recombinant 5-phosphatase I and pleckstrin in vitro show that they form a stoichiometric complex. A mutant form of pleckstrin, in which the serine and threonine residues that are phosphorylated by protein kinase C are substituted with glutamic acid (pseudophosphorylated pleckstrin), activates recombinant 5-phosphatase I 2-3-fold while native unphosphorylated pleckstrin does not stimulate the enzyme. Thus pleckstrin functions to terminate calcium signaling in platelets when it is phosphorylated by binding to and activating 5-phosphatase I.
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PMID:Phosphorylation of platelet pleckstrin activates inositol polyphosphate 5-phosphatase I. 899 61

Platelet-derived growth factor (PDGF) stimulates protein kinase D (PKD) in a time- and dose-dependent manner. We have used a series of PDGF receptor mutants that display a selective impairment of the binding of SH2-containing proteins (GTPase-activating protein, SHP-2, phospholipase Cgamma (PLCgamma), or phosphatidylinositol 3'-kinase (PI3K)) to show that Tyr-1021, the PLCgamma-binding site, is essential for PKD stimulation by PDGF in A431 cells. We next investigated whether any one of these four binding sites could mediate PKD activation in the absence of the other three sites. F5, a receptor mutant that lacks all four binding sites for GTPase-activating protein, PLCgamma, PI3K, and SHP-2, fails to activate PKD. A panel of single add-back mutants was used to investigate if any one of these four sites could restore signaling to PKD. Of the four sites, only the PLCgamma+ single add-back receptor restored PDGF-mediated activation of PKD, and only this add-back receptor produced diacylglycerol (DAG) in a PDGF-dependent manner. 1,2-Dioctanoyl-sn-glycerol, a membrane-permeant DAG analog, was found to be sufficient for activation of PKD. Taken together, these data indicate that PLCgamma activation is not only necessary, but also sufficient to mediate PDGF-induced PKD activation. Although the presence of a pleckstrin homology domain makes PKD a potential PI3K target, PKD was not stimulated by selective PI3K activation, and wortmannin, an inhibitor of PI3K, did not inhibit PDGF signaling to PKD. The activation of PKD by DAG or by the wild-type and PLCgamma+ add-back PDGF receptors was inhibited by GF109203X, suggesting a role for protein kinase C in the stimulation of PKD by PDGF. PDGF induced a time-dependent phosphorylation of PKD that closely correlated with activation. The PDGF-induced activation and phosphorylation of PKD were reversed by in vitro incubation of PKD with protein phosphatase 1 or 2A, indicating that PDGF signaling to PKD involves the Ser/Thr phosphorylation of PKD. Taken together, these results conclusively show that PDGF activates PKD through a pathway that involves activation of PLCgamma and, subsequently, protein kinase C.
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PMID:Platelet-derived growth factor stimulates protein kinase D through the activation of phospholipase Cgamma and protein kinase C. 950 12

Syk-family tyrosine kinases are essential for lymphocyte development and activation. Using a yeast two-hybrid screen to identify Syk kinases-interacting proteins (SKIPs), we isolated 3BP2, an Abl SH3-interacting protein of unknown function. 3BP2 was selectively expressed in hematopoietic/lymphoid tissues and bound via its SH2 domain activated Syk-family kinases in mammalian cells, including in antigen receptor-stimulated T cells. In addition to Zap-70, the 3BP2 SH2 domain associated in vitro with LAT, Grb2, PLCgamma1, and Cbl from activated T cell lysates. Transient 3BP2 overexpression induced transcriptional activation of the IL-2 promoter and its NFAT or AP-1 elements. This activity was dependent on the SH2 and pleckstrin-homology domains of 3BP2, and required functional Syk kinases, Ras, and calcineurin. Thus, 3BP2 is an important adaptor that may couple activated Zap-70/Syk to a LAT-containing signaling complex involved in TCR-mediated gene transcription.
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PMID:Adaptor function for the Syk kinases-interacting protein 3BP2 in IL-2 gene activation. 984 81

The Vav protooncogene is a multidomain protein involved in the regulation of IL-2 gene transcription in T cells and the development of cell-mediated killing by cytotoxic lymphocytes. We have investigated the differential roles that specific protein subdomains within the Vav protooncogene have in the development of these two distinct cellular processes. Interestingly, a calponin homology (CH) domain mutant of Vav (CH-) fails to enhance NF-AT/AP-1-mediated gene transcription but is still able to regulate the development of cell-mediated killing. The inability of the CH- mutant to enhance NF-AT/AP-1-mediated transcription appears to be secondary to defective intracellular calcium, because 1) the CH- mutant has significantly reduced TCR-initiated calcium signaling, and 2) treatment with the calcium ionophore ionomycin or cotransfection with activated calcineurin restores NF-AT/AP-1-mediated gene transcription. The pleckstrin homology (PH) domain of Vav has also been implicated in regulating Vav activation. We found that deletion of the PH domain of Vav yields a protein that can neither enhance gene transcription from the NF-AT/AP-1 reporter nor enhance TCR- or FcR-mediated killing. In contrast, the PH deletion mutant of Vav is able to regulate the development of natural cytotoxicity, indicating a functional dichotomy for the PH domain in the regulation of these two distinct forms of killing. Lastly, mutation of three tyrosines (Y142, Y160, and Y174) within the acidic domain of Vav has revealed a potential negative regulatory site. Replacement of all three tyrosines with phenylalanine results in a hyperactive protein that increases NF-AT/AP-1-mediated gene transcription and enhances cell-mediated cytotoxicity. Taken together, these data highlight the differential roles that specific subdomains of Vav have in controlling distinct cellular functions. More broadly, the data suggest that separate lymphocyte functions can potentially be modulated by domain-specific targeting of Vav and other critical intracellular signaling molecules.
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PMID:Specific subdomains of Vav differentially affect T cell and NK cell activation. 1075 87

The ppb1(+) gene encodes a fission yeast homologue of the mammalian calcineurin. We have recently shown that Ppb1 is essential for chloride ion homeostasis, and acts antagonistically with Pmk1 mitogen-activated protein kinase pathway. In an attempt to identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature, and isolated a mutant, its3-1. its3(+) was shown to be an essential gene encoding a functional homologue of phosphatidylinositol-4-phosphate 5-kinase (PI(4)P5K). The temperature upshift or addition of FK506 induced marked disorganization of actin patches and dramatic increase in the frequency of septation in the its3-1 mutants but not in the wild-type cells. Expression of a green fluorescent protein-tagged Its3 and the phospholipase Cdelta pleckstrin homology domain indicated plasma membrane localization of PI(4)P5K and phosphatidylinositol 4,5-bisphosphate. These green fluorescent protein-tagged proteins were concentrated at the septum of dividing cells, and the mutant Its3 was no longer localized to the plasma membrane. These data suggest that fission yeast PI(4)P5K Its3 functions coordinately with calcineurin and plays a key role in cytokinesis, and that the plasma membrane localization of Its3 is the crucial event in cytokinesis.
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PMID:Phosphatidylinositol 4-phosphate 5-kinase Its3 and calcineurin Ppb1 coordinately regulate cytokinesis in fission yeast. 1095 Sep 58

Analysis of protein-protein interaction is one of the powerful methods for elucidating the new functions of functionally unknown proteins. Using this approach, we isolated two proteins interacting with PRIP-1, which was isolated as a new Ins(1,4,5) P3 binding protein from brain. One was protein phosphatase 1 catalytic subunit (PP1c) and the other was GABARAP (GABAA-receptor-associated protein). The region of PRIP-1 responsible for their interaction was the site preceding to the pleckstrin homology domain of PRIP-1 for PP1c and the EF-hand motifs of PRIP-1 for GABARAP, which were determined by beta-galactosidase assay of yeast two-hybrid system. The association between PRIP-1 and PP1c was confirmed in vitro by a pull-down assay, a far-western assay, an immunoprecipitation analysis and a surface plasmon resonance analysis. The interaction of PRIP-1 with PP1c resulted in inhibition of the catalytic activity of PP1c in a PRIP-1 concentration-dependent manner. The association between PRIP-1 and GABARAP was also confirmed by a pull-down assay, and we found that PRIP-1 competitively inhibited the binding of the gamma 2 subunit of the GABAA receptor to GABARAP in vitro. Our electrophysiological and behavioral analysis of PRIP-1 knockout mice revealed that PRIP-1 is essential for the function of GABAA receptors, especially in response to the agents acting on the gamma 2 subunit.
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PMID:[The analysis of protein-protein interaction with special reference to PRIP-1]. 1197 30

Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha. Metabolic labeling with (32)P-orthophosphate in the transfected cells revealed that there is no major phosphorylation site other than the three residues in PKBalpha. PKBalpha activated by heat shock was more stable than the enzyme stimulated by PDGF in the cells, and PKBalpha recovered from heat-shocked cells was resistant to the protein phosphatase treatment, whereas the enzyme obtained from the growth-factor-stimulated cells was inactivated by dephosphorylation. Heat shock also enhanced the association of the PH-domain fragment to the full-length PKBalpha in the transfected cells. On the other hand, the PH-domain fragment of PKBalpha, which moves from the cytosol to the plasma membrane upon PDGF stimulation by the interaction with the phosphatidylinositol 3-kinase products, did not translocate but stayed in the cytosol in heat-shocked NIH 3T3 cells. Furthermore, PKBalpha was associated with the nuclear region in heat-shocked cells, which is not observed in growth-factor-stimulated cells. These results indicate that heat shock induces the conformational change of PKBalpha that accompanies the protein complex formation and perinuculear/nuclear localization of the enzyme, to generate an active form by a mechanism distinct from that in the growth-factor-signaling pathway.
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PMID:Distinct activation mechanisms of protein kinase B by growth-factor stimulation and heat-shock treatment. 1506 72

PDK1 and PKB/Akt have a pleckstrin homology (PH) domain at the C-terminus and N-terminus, respectively, which stabilizes an unphosphorylated, autoinhibited conformation. Binding of the PH domain to a phospholipid second messenger causes relief of autoinhibition, which results in kinase phosphorylation and activation. Baculovirus-mediated expression in Sf9 insect cells of both His(6)-PDK1 and His(6)-PKBbeta/Akt2 were optimized, which significantly improved the yields (5-fold) of the affinity purified enzymes over previously reported values. Isoelectric focusing (IEF) and Western analyses indicated that the apparent V(max)=192+/-13 U/mg and K(m) (PDK-Tide)=55+/-10 microM of purified His(6)-PDK1 results from a mixture of at least three different phospho-specific isoforms (pI values of 6.8, 6.5, and 6.4). A purely unphosphorylated isoform of His(6)-PDK1 (pI=6.8) was generated by treatment with lambda protein phosphatase (lambdaPP), which decreased V(max) to 2.4+/-0.4 U/mg and increased K(m) (PDK-Tide) to 217+/-61 microM. Isoelectric focusing and Western analyses indicated that the apparent V(max)=0.21+/-0.03 U/mg and K(m) (Crosstide)=87+/-30 microM of purified His(6)-PKBbeta/Akt2 results from a mixture of the enzyme monophosphorylated either at Ser-474 ( approximately 90%) or at Thr-309 ( approximately 10%). A purely unphosphorylated isoform of His(6)-PKBbeta/Akt2 (pI=6.4) was generated by treatment with lambdaPP, which decreased V(max) approximately 2-fold. The optimization of high-level production and detailed characterization of purified and lambdaPP-treated His(6)-PDK1 and His(6)-PKBbeta/Akt2 will facilitate detailed structural and kinetic studies aimed at understanding the mechanism of second messenger-induced activation.
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PMID:Improved yields for baculovirus-mediated expression of human His(6)-PDK1 and His(6)-PKBbeta/Akt2 and characterization of phospho-specific isoforms for design of inhibitors that stabilize inactive conformations. 1608 96

Investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogs has led to the isolation of a novel binding protein with a molecular size of 130 kDa, characterized as a molecule with similar domain organization to phospholipase C-d1 (PLC-d1) but lacking the enzymatic activity. An isoform of the molecule was subsequently identified, and these molecules have been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind Ins(1,4,5)P3 via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P3-mediated Ca2+ signaling was examined using COS-1 cells overexpressing PRIP-1 and cultured neurons prepared from PRIP-1 knock-out mice. Yeast two hybrid screening of a brain cDNA library using a unique N-terminus as bait identified GABARAP (GABAA receptor associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible involvement of PRIP in GABAA receptor signaling. For this purpose PRIP knock-out mice were analyzed for GABAA receptor function in relation to the action of benzodiazepines from the electrophysiological and behavioral aspects. During the course of these experiments we found that PRIP also binds to the b-subunit of GABAA receptors and PP2A (protein phosphtase 2A). Here, we summarize how PRIP is involved in Ins(1,4,5)P3-mediated Ca2+ signaling and GABAA receptor signaling based on the characteristics of binding molecules.
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PMID:PRIP, a novel Ins(1,4,5)P3 binding protein, functional significance in Ca2+ signaling and extension to neuroscience and beyond. 1640 43


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