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)

Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation of N-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation.
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PMID:Phosphorylation of protein phosphatase inhibitor-1 by Cdk5. 1127 34

Na(+)/H(+) exchanger isoform-1 (NHE1), the ubiquitous form of the Na(+)/H(+) exchanger, has increased activity in hypertensive patients and in animal models of hypertension. Furthermore, NHE1 is activated in cells stimulated with growth factors. We showed previously that activation of the exchanger is dependent on phosphorylation of serine 703 (Ser(P)(703)) by p90 ribosomal S6 kinase (RSK). Because the NHE1 sequence at Ser(P)(703) (RIGSDP) is similar to a consensus sequence (RSXSXP) specific for 14-3-3 ligands, we evaluated whether serum stimulated 14-3-3 binding to NHE1. Five different GST-NHE1 fusion proteins spanning amino acids 515-815 were phosphorylated by RSK and used as ligands in a far Western analysis; only those containing Ser(P)(703) exhibited high affinity 14-3-3 binding. In PS127A cells (NHE1-overexpressing Chinese hamster fibroblasts) stimulated with 20% serum, NHE1 co-precipitation with GST-14-3-3 fusion protein increased at 5 min (5.2 +/- 0.4-fold versus control; p < 0.01) and persisted at 40 min (3.9 +/- 0.3-fold; p < 0.01). We confirmed that binding occurs at the RIGSDP motif using PS120 (NHE1 null) cells transfected with S703A-NHE1 or P705A-NHE1 (based on data indicating that 14-3-3 binding requires phosphoserine and +2 proline). Serum failed to stimulate association of 14-3-3 with these mutants. A GST-NHE1 fusion protein was phosphorylated by RSK and used as a ligand to assess the effect of 14-3-3 on protein phosphatase 1-mediated dephosphorylation of Ser(P)(703). GST-14-3-3 limited dephosphorylation (66% of initial state at 60 min) compared with GST alone (27% of initial state; p < 0.01). The protective effect of GST-14-3-3 was lost in the GST-NHE1 P705A mutant. Finally, the base-line rate of pH recovery in acid-loaded cells was equal in unstimulated cells expressing wild-type or P705A-NHE1. However, activation of NHE1 by serum was dramatically inhibited in cells expressing P705A-NHE1 compared with wild-type (0.13 +/- 0.02 versus 0.48 +/- 0.06 mmol of H(+)/min/liter, p < 0.01). These data suggest that 14-3-3 binding to NHE1 participates in serum-stimulated exchanger activation, a new function for 14-3-3.
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PMID:14-3-3 Binding to Na+/H+ exchanger isoform-1 is associated with serum-dependent activation of Na+/H+ exchange. 1127 64

Directed neuronal, astroglial, and oligodendroglial cell migrations comprise a prominent feature of mammalian brain development. Because molecular motor proteins have been implicated in a wide spectrum of processes associated with cell motility, we initiated studies to define the pool of myosins in migrating cerebellar granule neurons and type-1 neocortical astrocytes. Our analyses identified two isoforms of a novel unconventional myosin, which we have cloned, sequenced, and designated myr 8a and 8b (eighth unconventional myosin from rat). Phylogenetic analysis indicates that myr 8 myosins comprise a new class of myosins, which we have designated class XVI. The head domain contains a large N-terminal extension composed of multiple ankyrin repeats, which are implicated in mediating an association with the protein phosphatase 1 (PP1) catalytic subunits 1alpha and 1gamma. The motor domain is followed by a single putative light-chain binding domain. The tail domain of myr 8a is comparatively short with a net positive charge, whereas the tail domain of myr 8b is extended, bears an overall neutral charge, and reveals several stretches of poly-proline residues. Neither the myr 8a nor the myr 8b sequence reveals alpha-helical coiled-coil motifs, suggesting that these myosins exist as monomers. Both immunoblot and Northern blot analyses indicate that myr 8b is the predominant isoform expressed in brain, principally at developmental time periods. The structural features and restricted expression patterns suggest that members of this novel class of unconventional myosins comprise a mechanism to target selectively the protein phosphatase 1 catalytic subunits 1alpha and/or 1gamma in developing brain.
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PMID:Myr 8, a novel unconventional myosin expressed during brain development associates with the protein phosphatase catalytic subunits 1alpha and 1gamma1. 1158 69

The nuclear localization and transcriptional activity of the NF-ATc family of transcription factors, essential to many developmental, differentiation, and adaptation processes, are determined by the opposing activities of the phosphatase calcineurin, which promotes nuclear accumulation of NF-ATc, and several kinases, which promote cytoplasmic accumulation. Many reports suggest that protein kinase A (PKA) negatively modulates calcineurin-mediated NF-ATc activation. Here we show that overexpression of PKA causes phosphorylation and cytoplasmic accumulation of NF-ATc1 in direct opposition to calcineurin by phosphorylating Ser-245, Ser-269, and Ser-294 in the conserved serine-proline repeat domain, and that mutation of these serines blocks the effect of PKA. Activation of endogenous PKA is similarly able to promote phosphorylation of these sites on NF-ATc1 in two lymphoid cell lines. We further show that a complete block of NF-ATc1 nuclear localization by PKA requires a second kinase activity that can be supplied by glycogen synthase kinase-3 (GSK-3), and that mutation of either the PKA phosphorylation sites or the upstream GSK-3 sites prevents the effect of PKA. Thus, we propose that PKA functions cooperatively as a priming kinase for further phosphorylation by GSK-3 to oppose calcineurin-mediated nuclear accumulation and transcriptional activity of NF-ATc1 and that, through this mechanism, PKA may be an important modulator of many NF-ATc-dependent processes.
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PMID:Protein kinase A negatively modulates the nuclear accumulation of NF-ATc1 by priming for subsequent phosphorylation by glycogen synthase kinase-3. 1235 31

Although the phosphorylation-dependent activation of the Na-K-Cl cotransporter (NKCC1) has been previously well documented, the identity of the kinase(s) responsible for this regulation has proven elusive. Recently, Piechotta et al. (Piechotta, K., Lu, J., and Delpire, E. (2002) J. Biol. Chem. 277, 50812-50819) reported the binding of PASK (also referred as SPAK (STE20/SPS1-related proline-alanine-rich kinase)) and OSR1 (oxidative stress response kinase) to cation-chloride cotransporters KCC3, NKCC1, and NKCC2. In this report, we show that overexpression of a kinase inactive, dominant negative (DN) PASK mutant drastically reduces both shark (60 +/- 5%) and human (80 +/- 3%) NKCC1 activation. Overexpression of wild type PASK causes a small (sNKCC1 22 +/- 8% p < 0.05, hNKCC1 12 +/- 3% p < 0.01) but significant increase in shark and human cotransporter activity in HEK cells. Importantly, DNPASK also inhibits the phosphorylation of two threonines, contained in the previously described N-terminal regulatory domain. We additionally show the near complete restoration of NKCC1 activity in the presence of the protein phosphatase type 1 inhibitor calyculin A, demonstrating that DNPASK inhibition results from an alteration in kinase/phosphatase dynamics rather than from a decrease in functional cotransporter expression. Coimmunoprecipitation assays confirm PASK binding to NKCC1 in transfected HEK cells and further suggest that this binding is not a regulated event; neither PASK nor NKCC1 activity affects the association. In cells preloaded with 32Pi, the phosphorylation of PASK, but not DNPASK, coincides with that of NKCC1 and increases 5.5 +/- 0.36-fold in low [Cl]e. These data conclusively link PASK with the phosphorylation and activation of NKCC1.
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PMID:PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1). 1274 Mar 79

Cyclophilin A (CypA/Ppia) is a peptidyl-prolyl isomerase (PPIase) that binds the immunosuppressive drug cyclosporine. The resulting complex blocks T cell function by inhibiting the calcium-dependent phosphatase calcineurin. To identify the native function of CypA, long suspected of regulating signal transduction, we generated mice lacking the Ppia gene. These animals develop allergic disease, with elevated IgE and tissue infiltration by mast cells and eosinophils, that is driven by CD4+ T helper type II (Th2) cytokines. Ppia(-/-) Th2 cells were hypersensitive to TCR stimulation, a phenotype consistent with increased activity of Itk, a Tec family tyrosine kinase crucial for Th2 responses. CypA bound Itk via the PPIase active site. Mutation of a conformationally heterogeneous proline in the SH2 domain of Itk disrupted interaction with CypA and specifically increased Th2 cytokine production from wild-type CD4+ T cells. Thus, CypA inhibits CD4+ T cell signal transduction in the absence of cyclosporine via a regulatory proline residue in Itk.
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PMID:Cyclophilin A regulates TCR signal strength in CD4+ T cells via a proline-directed conformational switch in Itk. 1530

Cyclophilin (CyP) is a cytosolic receptor of immunosuppressive drug cyclosporin A (CsA). The binary complex of CyP-CsA inhibits the activity of Ca2+/calmodulin dependent serine/threonine calcineurin (CN). The inhibition of CN in turn disables the transcription activity of nuclear factor of activated T cell, thus suppressing the T cell activation and cardiac hypertrophy. CyP is also an enzyme catalyzing peptidyl-prolyl cis-trans isomerization and serves as a molecular chaperone in various biological processes. For example, CyPA is involved in the assembly/deassembly of HIV-1 virion and is required for the full infectious activity of HIV-1. However, the in vivo function of CyP remains a mystery. This review will describe the three-dimensional structures of CyPs and its partners and discuss the structural clues to understanding the CyP functions in biological processes. The structures of CyP in complex with proline-containing peptides provided insight into the mechanism of peptidyl-prolyl cis-trans isomerization. The structures of CyPA in complex with HIV-1 capsid protein and its peptides revealed details of interactions of CyP with HIV-1 capsid protein, thus providing a guideline for design of anti-HIV drugs. The rearrangement of two tetratricopeptide repeats of the, large, cyclophilin CyP40 into a long helix under the crystallization conditions might be biologically relevant to the CyP40 function in the hsp90 molecular chaperone system. The structures of the binary CyPA-CsA and ternary CN-CyPA-CsA complexes showed how CsA binds to its receptors and therefore provide a template for design of new immunosuppressive drugs.
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PMID:Crystal structures of cyclophilin and its partners. 1535 87

The Raf-1 kinase is an important signaling molecule, functioning in the Ras pathway to transmit mitogenic, differentiative, and oncogenic signals to the downstream kinases MEK and ERK. Because of its integral role in cell signaling, Raf-1 activity must be precisely controlled. Previous studies have shown that phosphorylation is required for Raf-1 activation, and here, we identify six phosphorylation sites that contribute to the downregulation of Raf-1 after mitogen stimulation. Five of the identified sites are proline-directed targets of activated ERK, and phosphorylation of all six sites requires MEK signaling, indicating a negative feedback mechanism. Hyperphosphorylation of these six sites inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to additional stimuli. The hyperphosphorylated/desensitized Raf-1 is subsequently dephosphorylated and returned to a signaling-competent state through interactions with the protein phosphatase PP2A and the prolyl isomerase Pin1. These findings elucidate a critical Raf-1 regulatory mechanism that contributes to the sensitive, temporal modulation of Ras signaling.
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PMID:Regulation of Raf-1 by direct feedback phosphorylation. 1566 84

Activity of Na+-K+-2Cl- co-transport (NKCC1) in epithelia is thought to be highly regulated through phosphorylation and dephosphorylation of the transporter. Previous functional studies from this laboratory suggested a role for protein phosphatase 2A (PP2A) as a serine/threonine protein phosphatase involved in the regulation of mammalian tracheal epithelial NKCC1. We expand on these studies to characterize serine/threonine protein phosphatase(s) necessary for regulation of NKCC1 function and the interaction of the phosphatase(s) with proteins associated with NKCC1. NKCC1 activity was measured as bumetanide-sensitive 86Rb uptake or basolateral to apical 86Rb flux in primary cultures of human tracheal epithelial cells or in Calu-3 airway epithelial cells grown on Transwell filter inserts. Preincubation with 0.1 nm okadaic acid, a PP2A >> phosphatase 1 (PP1) inhibitor, increased NKCC1 activity 3.5-fold in human tracheal epithelial cells and 4.1-fold in Calu-3 cells. Calyculin, a PP1 >> PP2A inhibitor, did not alter NKCC1 activity or percent bumetanide-sensitive flux. The effect of OA was dose-dependent with an IC50 of 0.4 nm. The alpha1-adrenergic agonist methoxamine increased NKCC1 activity and transiently increased PP2A activity 3.8-fold but did not alter PP1 activity. OA augmented methoxamine-dependent stimulation of NKCC1 activity. PP1, PP2A, and PP2C but not PP2B were detected in lysates from Calu-3 cells by immunoblot analysis. PP1 was not detected in immunoprecipitates of NKCC1 and vice versa. PP2A co-immunoprecipitated with NKCC1 and protein kinase C-delta (PKC-delta) and was pulled down by a recombinant N terminus of NKCC1 consisting of amino acids 1-286. One novel finding is co-precipitation of STE20-related proline-alanine-rich kinase, a regulatory kinase for NKCC1, with PP2A and PKC-delta. The results suggest a model of actin serving as a scaffold for binding and association of PKC-delta, PP2A, and STE20-related proline-alanine-rich kinase. The role of the complex of serine/threonine protein kinases and a protein phosphatase is probably the maintenance of optimal phosphorylation of NKCC1 coincident with its physiological function in epithelial absorption and secretion.
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PMID:Role for protein phosphatase 2A in the regulation of Calu-3 epithelial Na+-K+-2Cl-, type 1 co-transport function. 1589 83

Proline oxidase is a p53-induced redox gene that can generate reactive oxygen species (ROS) and mediate apoptosis in tumor cells. We report that proline oxidase is a downstream effector in p53-mediated activation of the calcium/calmodulin-dependent phosphatase calcineurin in lung, renal, colon, and ovarian carcinoma cells. The activation of calcineurin by p53 and proline oxidase was detected by activation of the nuclear factor of activated T cells (NFAT), an established indicator of activated calcineurin. Both proline oxidase- and p53-induced activation of NFAT were sensitive to the calcineurin inhibitors cyclosporin A and FK-506, to scavengers of ROS, and to inhibitors of calcium mobilization. A proline oxidase antisense vector suppressed the ability of p53 to up-regulate proline oxidase, activate calcineurin, and induce apoptosis. Moreover, two renal carcinoma-derived mutant p53 proteins were deficient in inducing proline oxidase expression and in activating calcineurin. Inhibitors of calcineurin and calcium mobilization abolished proline oxidase-mediated apoptosis and reduced p53-induced apoptosis. Treatment of colon and ovarian carcinoma cells with the anticancer genotoxic agent etoposide up-regulated both p53 and proline oxidase, activated calcineurin, and induced apoptosis. The etoposide-mediated activation of calcineurin and induction of apoptosis was markedly suppressed by FK-506 calcineurin inhibitor. We propose that proline oxidase mediates apoptosis through the generation of proline-dependent ROS, which then mobilize calcium and activate calcineurin. The activation of calcineurin-regulated transcription factor pathways by proline oxidase might affect gene expression events important to p53 regulation of cell growth and apoptosis.
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PMID:The p53-induced gene-6 (proline oxidase) mediates apoptosis through a calcineurin-dependent pathway. 1591 62


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