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)

Phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 at Ser(845) enhances AMPA channel activity. This study demonstrates that Ser(845) is rapidly dephosphorylated upon AMPA receptor activation in nucleus accumbens slices. AMPA-induced dephosphorylation at Ser(845) was blocked by CNQX, an AMPA receptor antagonist, by nifedipine, an L-type Ca(2+) channel antagonist, or by cyclosporin A, a calcineurin inhibitor. N-methyl-D-aspartate (NMDA) treatment also decreased phosphorylation of Ser(845), an effect that was blocked by MK-801, an NMDA receptor antagonist, but not by nifedipine. Accumbens neurons are enriched for dopamine- and cyclic AMP (cAMP)-regulated phosphoprotein, Mr 32,000 (DARPP-32), a potent inhibitor of protein phosphatase 1 (PP1) when phosphorylated by PKA (at Thr(34)). We tested the hypothesis that the AMPA/KA or NMDA-stimulated dephosphorylation of DARPP-32 via calcineurin, leading to increased PP1 activity and dephosphorylation of GluR1. AMPA or NMDA treatment decreased phospho-Thr(34)-DARPP-32 levels, effects that were blocked by receptor antagonists, or cyclosporin A. However, dephosphorylation of Ser(845) mediated by AMPA or NMDA receptors was unaffected in DARPP-32/inhibitor-1 knockout mice. These data suggest that AMPA- or NMDA-induced dephosphorylation of GluR1 at Ser(845) occurs by a mechanism that is independent of DARPP-32 and PP1, but involves activation of calcineurin. Thus, Ca(2+)-dependent dephosphorylation of GluR1 may serve as a negative feedback mechanism for the regulation of AMPA receptor activity in neurons.
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PMID:Regulation of AMPA receptor dephosphorylation by glutamate receptor agonists. 1452 9

The HePTP (haematopoietic protein tyrosine phosphatase) is a negative regulator of the ERK2 (extracellular signal-regulated protein kinase 2) and p38 MAP kinases (mitogen-activated protein kinases) in T-cells. This inhibitory function requires a physical association of HePTP through an N-terminal KIM (kinase-interaction motif) with ERK and p38. We previously reported that PKA (cAMP-dependent protein kinase) phosphorylates Ser-23 within the KIM of HePTP, resulting in dissociation of HePTP from ERK2. Here we follow the phosphorylation of this site in intact T-cells. We find that HePTP is phosphorylated at Ser-23 in resting T-cells and that this phosphorylation increases upon treatment of the cells with agents that elevate intracellular cAMP, such as prostaglandin E2. HePTP phosphorylation occurred at discrete regions at the cell surface. Phosphorylation was reduced by inhibitors of PKA and increased by inhibitors of protein phosphatases PP1 and PP2A, but not by inhibitors of calcineurin. In vitro, PP1 efficiently dephosphorylated HePTP at Ser-23, while PP2A was much less efficient. Activation of PP1 by treatment of the cells with ceramide suppressed Ser-23 phosphorylation, as did transfection of the catalytic subunit of PP1. Phosphorylation at Ser-23 is also increased in a transient manner upon T-cell antigen receptor ligation. In contrast, treatment of cells with phorbol ester had no effect on HePTP phosphorylation at Ser-23. We conclude from these results that HePTP is under continuous control by PKA and a serine-specific phosphatase, probably PP1, in T-cells and that this basal phosphorylation at Ser-23 can rapidly change in response to external stimuli. This, in turn, will affect the ability of HePTP to inhibit the ERK and p38 MAP kinases.
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PMID:Haematopoietic protein tyrosine phosphatase (HePTP) phosphorylation by cAMP-dependent protein kinase in T-cells: dynamics and subcellular location. 1461 83

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.
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PMID:PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation. 1463 64

Expression of recombinant PP1 isoforms with fully authentic properties has proven to be a challenge for several laboratories. In order to circumvent this technical limitation in the investigation of isoform-specific roles for PP1, methods have been developed to analyze specific properties of native PP1 isoforms. The well-documented method of ethanol precipitation of tissue extracts has been used to dissociate phosphatase catalytic subunits from their endogenous regulatory subunits and other cellular proteins. Although very low levels of PP1 and PP2A regulatory subunits are sometimes detected in PPC preparations, they are not associated with their respective catalytic subunits because they do not copurify with the catalytic subunits on microcystin-Sepharose (Bauman & Colbran, not shown). Thus, the PPC preparation represents a mixture of native monomeric phosphatase catalytic subunits (including PP1 isoforms, PP2AC, PP4C, and PP6C) that can be used to analyze their interactions with other proteins. The methods described in this report rely on the availability of highly specific antibodies to PP1 isoforms. The sheep antibodies have previously proven effective for immunoblotting and immunoprecipitation, whereas rabbit antibodies have also been used for immunocytochemistry. This paper documents the use of these antibodies in Far-Western overlay and glutathione-agarose cosedimentation assays to investigate interactions of specific PP1 isoforms with recombinant fragments of PP1-targeting subunits (spinophilin, neurabin and GM). Moreover, covalent coupling of affinity-purified sheep antibodies to agarose provided a means for the immuno-isolation of PP1 beta and PP1 gamma 1 from the PPC preparation. Active catalytic subunits are recovered from the affinity resin using chaotropic agents, permitting for the first time the assessment of the effects of specific targeting subunits on activities of individual native PP1 isoforms. These methods have been used successfully to demonstrate that some PP1-interacting proteins discriminate among the isoforms. The isoform inhibition assays provide a measure of the binding equilibrium in the milieu of the phosphatase assay. For example, while some PP1-binding proteins inhibit native PP1 beta and native PP1 gamma 1 with equivalent potency (e.g., PKA-phosphorylated inhibitor-1), spinophilin, neurabin and GM differentiate between these two isoforms; spinophilin and neurabin fragments inhibit native PP1 gamma 1 approximately 20-fold more potently than they inhibit native PP1 beta (Fig. 4), whereas GM inhibits native PP1 beta more potently than native PP1 gamma 1 (not shown). Moreover, the activity of native PP1 gamma 1 is approximately 100-fold more sensitive to neurabin and spinophilin than is the activity of bacterially-expressed recombinant PP1 gamma 1 (Fig. 4). The interpretation of these inhibition assays is consistent with data obtained in Far-Western overlay (Fig. 2) and glutathione-agarose cosedimentation assays (Fig. 3), which assess more stable interactions of PP1 isoforms. Thus, spinophilin and neurabin selectively bind PP1 gamma 1 over PP1 beta, whereas GM is highly selective for PP1 beta. These data are consistent with previous experiments that showed spinophilin and neurabin are present in PP1 gamma 1 complexes in brain extracts, but not in PP1 beta complexes. Moreover, only PP1 beta has been identified in complexes with GM in muscle extracts, although these data did not exclude the possibility that other isoforms were also present. Presumably, these isoform-selective interactions confer different functions on PP1. In summary, we have developed methods that should prove useful in defining the isoform-selectivity of other PP1-targeting subunits. Moreover, these methods may be employed to identify domains in PP1-interacting proteins that confer isoform specificity. Similar strategies may also be used to explore interactions of protein phosphatase catalytic subunits with other proteins.
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PMID:Analysis of specific interactions of native protein phosphatase 1 isoforms with targeting subunits. 1467 48

In boar spermatozoa, the capacitating agent bicarbonate has been shown to induce rapid changes both in plasma membrane lipid architecture and in motility; in each case, a PKA-dependent pathway is involved. Early bicarbonate-induced changes in protein phosphorylation were probed using a commercial antibody against the phosphorylated form of the consensus substrate site for cyclic AMP-dependent protein kinase. The antibody detected relatively few bands in sperm extracts, of which only a small number showed incubation-dependent changes. While the quantitative response varied between boar ejaculates, in general terms bicarbonate induced phosphorylation increases in bands of 96, 64, and 59 kDa within 80 sec. The changes reached a maximum after about 160 sec, declined somewhat thereafter, and then increased again slowly as incubation progressed further (up to 21 min). The bicarbonate-induced increases were strongly dependent on the presence of BSA in the incubation medium. They were inhibited by H89 (PKA inhibitor) but not by GF (PKC inhibitor), and were enhanced by papaverine (phosphodiesterase inhibitor) and by calyculin (protein phosphatase inhibitor). The cyclic AMP analogue cBIMPS was able to mimic bicarbonate action though its effect was less dramatic. Stearated Ht31, a permeable inhibitor of PKA's binding to A-kinase anchoring protein, did not affect either the intensity or the specificity of the bicarbonate-induced phosphorylation changes, though it blocked motility entirely. Immunocytochemical studies revealed marked bicarbonate-dependent phosphorylation changes in the post-acrosomal region of the head and in the neck, midpiece, and anterior regions of the tail. Fractionation of stimulated spermatozoa showed that all bands detectable with the antibody were bound to heads and to midpieces and associated large tail fragments; no bands were detected in either small tail or membrane fragments or in the cytoplasmic fraction. Differential extraction of the midpiece/large tail fraction revealed two protein bands with closely similar electrophoretic mobilities to the 96- and 59-kDa phosphorylated bands; MALDI-TOF analyses of these bands revealed both to be members of the Odf2 family.
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PMID:Rapid PKA-catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate. 1473 95

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.
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PMID:Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25. 1476 70

The CREB family of proteins are critical mediators of gene expression in response to extracellular signals and are essential regulators of adaptive behavior and long-term memory formation. The TORC proteins were recently described as potent CREB coactivators, but their role in regulation of CREB activity remained unknown. TORC proteins were found to be exported from the nucleus in a CRM1-dependent fashion. A high-throughput microscopy-based screen was developed to identify genes and pathways capable of inducing nuclear TORC accumulation. Expression of the catalytic subunit of PKA and the calcium channel TRPV6 relocalized TORC1 to the nucleus. Nuclear accumulation of the three human TORC proteins was induced by increasing intracellular cAMP or calcium levels. TORC1 and TORC2 translocation in response to calcium, but not cAMP, was mediated by calcineurin, and TORC1 was shown to be directly dephosphorylated by calcineurin. TORC function was shown to be essential for CRE-mediated gene expression induced by cAMP, calcium, or GPCR activation, and nuclear transport of TORC1 was sufficient to activate CRE-dependent transcription. Drosophila TORC was also shown to translocate in response to calcineurin activation in vivo. Thus, TORC nuclear translocation is an essential, conserved step in activation of cAMP-responsive genes.
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PMID:Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins. 1558 60

Receptor-mediated dopamine (DA) modulation of neuronal excitability in the nucleus accumbens (NAc) has been shown to be critically involved in drug addiction and a variety of brain diseases. However, the mechanisms underlying the physiological or pathological molecular process of DA modulation remain largely elusive. Here, we demonstrate that stimulation of DA D2 class receptors (D2R) enhanced voltage-sensitive sodium currents (VSSCs, I(Na)) in freshly dissociated NAc neurons via suppressing tonic activity of the cyclic AMP/PKA cascade and facilitating intracellular Ca2+ signaling. D2R-mediated I(Na) enhancement depended on activation of G(i/o) proteins and was mimicked by direct inhibition of PKA. Furthermore, increasing free [Ca2+]in by activating inositol 1,4,5-triphosphate receptors (IP3Rs), blocking Ca2+ reuptake, or adding buffered Ca2+, all enhanced I(Na). Under these circumstances, D2R-mediated I(Na) enhancement was occluded. In contrast, D2R-mediated I(Na) enhancement was blocked by inhibition of IP3Rs, chelation of free Ca2+, or inhibition of Ca2(+)/calmodulin-activated calcineurin (CaN), but not by inhibition of phospholipase C (PLC). Although stimulation of muscarinic cholinergic receptors (mAChRs) also increased I(Na), this action was blocked by PLC inhibitors. Our findings indicate that D2Rs mediate an enhancement of VSSCs in NAc neurons, in which cytosolic free Ca2+ plays a crucial role. Our results also suggest that D2R-mediated reduction in tonic PKA activity may increase free [Ca2+]in, primarily via disinhibition of IP3Rs. IP3R activation then facilitates Ca2+ signaling and subsequently enhances VSSCs via decreasing PKA-induced phosphorylation and increasing CaN-induced dephosphorylation of Na+ channels. This study provides insight into the complex and dynamic role of D2Rs in the NAc.
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PMID:Dopamine D2 receptor-activated Ca2+ signaling modulates voltage-sensitive sodium currents in rat nucleus accumbens neurons. 1559 Jul 33

The abnormal hyperphosphorylation of tau protein is one of the hallmarks of Alzheimer disease and other tauopathies; as yet the exact role of various tau kinases in this pathology is not fully understood. Here, we show that injection of isoproterenol, an activator of cAMP-dependent kinase (PKA), into rat hippocampus bilaterally results in the activation of PKA, calcium/calmodulin-dependent kinase II and cyclin-dependent kinase-5, inhibition of protein phosphatase-2A, hyperphosphorylation of tau at several Alzheimer-like epitopes and a disturbance of spatial memory retention 48 h after the drug injection. These findings suggest the involvement of PKA and PKA-mediated signaling pathway in the Alzheimer-like tau hyperphosphorylation and memory impairment.
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PMID:Bilateral injection of isoproterenol into hippocampus induces Alzheimer-like hyperphosphorylation of tau and spatial memory deficit in rat. 1562 Jul 22

The potassium channel KCNQ4, expressed in the mammalian cochlea, has been associated tentatively with an outer hair cell (OHC) potassium current, I(K,n), a current distinguished by an activation curve shifted to exceptionally negative potentials. Using CHO cells as a mammalian expression system, we have examined the properties of KCNQ4 channels under different phosphorylation conditions. The expressed current showed the typical KCNQ4 voltage-dependence, with a voltage for half-maximal activation (V(1/2)) of -25 mV, and was blocked almost completely by 200 microM linopirdine. Application of 8-bromo-cAMP or the catalytic sub-unit of PKA shifted V(1/2) by approximately -10 and -20 mV, respectively. Co-expression of KCNQ4 and prestin, the OHC motor protein, altered the voltage activation by a further -15 mV. Currents recorded with less than 1 nM Ca(2+) in the pipette ran down slowly (12% over 5 min). Buffering the pipette Ca(2+) to 100 nM increased the run-down rate sevenfold. Exogenous PKA in the pipette prevented the effect of elevated [Ca(2+)](i) on run-down. Inhibition of the calcium binding proteins calmodulin or calcineurin by W-7 or cyclosporin A, respectively, also prevented the calcium-dependent rapid run-down. We suggest that KCNQ4 phosphorylation via PKA and coupling to a complex that may include prestin can lead to the negative activation and the negative resting potential found in adult OHCs.
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PMID:Regulation of the voltage-gated potassium channel KCNQ4 in the auditory pathway. 1566 Feb 59


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