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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Uptake by the dopamine transporter (DAT) is the primary pathway for the clearance of extracellular dopamine (DA) and consequently for regulating the magnitude and duration of dopaminergic signaling. Amphetamine (AMPH) has been shown to decrease simultaneously DAT cell-surface expression and [(3)H]DA uptake. We have shown that insulin and its subsequent signaling through the phosphatidylinositol 3-kinase (PI3K)-dependent pathway oppose this effect of AMPH by promoting increased cell-surface expression. Here, we used human embryonic kidney 293 cells stably expressing the human DAT (hDAT cells) to investigate the downstream cellular components important for this effect of insulin. Akt is a protein kinase effector immediately downstream of PI3K. Both overexpression of a dominant-negative mutant of Akt (K179R) and the addition of 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML9), a pharmacological inhibitor of Akt, decreased cell-surface expression of DAT, suggesting a role of basal Akt signaling in the homoeostasis of DAT. Moreover, expression of a constitutively active Akt mutant reduced the ability of AMPH to decrease hDAT cell-surface expression as well as [(3)H]DA uptake. In contrast, overexpression of K179R blocked the ability of insulin to oppose AMPH-induced reduction of hDAT cell-surface expression and [(3)H]DA uptake, as did ML9. Our data demonstrate that hDAT cell-surface expression is regulated by the insulin signaling pathway and that Akt plays a key role in the hormonal modulation of AMPH-induced hDAT trafficking and in the regulation of basal hDAT cell-surface expression.
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PMID:Akt is essential for insulin modulation of amphetamine-induced human dopamine transporter cell-surface redistribution. 1579 21

6-Hydroxydopamine is a neurotoxin commonly used to lesion dopaminergic pathways and generate experimental models for Parkinson disease, however, the cellular mechanism of 6-hydroxydopamine-induced neurodegeneration is not well defined. In this study we have explored how 6-hydroxydopamine neurotoxicity is initiated. We have also investigated downstream signaling pathways activated in response to 6-hydroxydopamine, using a neuronal-like, catecholaminergic cell line (PC12 cells) as an in vitro model system. We have shown that 6-hydroxydopamine neurotoxicity is initiated via extracellular auto-oxidation and the induction of oxidative stress from the oxidative products generated. Neurotoxicity is completely attenuated by preincubation with catalase, suggesting that hydrogen peroxide, at least in part, evokes neuronal cell death in this model. 6-Hydroxydopamine does not initiate toxicity by dopamine transporter-mediated uptake into PC12 cells, because both GBR-12909 and nisoxetine (inhibitors of dopamine and noradrenaline transporters, respectively) failed to reduce toxicity. 6-Hydroxydopamine has previously been shown to induce both apoptotic and necrotic cell-death mechanisms. In this study oxidative stress initiated by 6-hydroxydopamine caused mitochondrial dysfunction, activation of caspases 3/7, nuclear fragmentation, and apoptosis. We have shown that, in this model, proteolytic activation of the proapoptotic protein kinase Cdelta (PKCdelta) is a key mediator of 6-hydroxydopamine-induced cell death. 6-Hydroxydopamine induces caspase 3-dependent cleavage of full-length PKCdelta (79 kDa) to yield a catalytic fragment (41 kDa). Inhibition of PKCdelta (with rottlerin or via RNA interference-mediated gene suppression) ameliorates the neurotoxicity evoked by 6-hydroxydopamine, implicating this kinase in 6-hydroxydopamine-induced neurotoxicity and Parkinsonian neurodegeneration.
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PMID:6-hydroxydopamine-induced apoptosis is mediated via extracellular auto-oxidation and caspase 3-dependent activation of protein kinase Cdelta. 1636 Dec 58

Recent investigations have shown that three major striatal-signaling pathways (protein kinase A/DARPP-32, Akt/glycogen synthase kinase 3, and ERK) are involved in the regulation of locomotor activity by the monoaminergic neurotransmitter dopamine. Here we used dopamine transporter knock-out mice to examine which particular changes in the regulation of these cell signaling mechanisms are associated with distinct behavioral responses to psychostimulants. In normal animals, amphetamine and methylphenidate increase extracellular levels of dopamine, leading to an enhancement of locomotor activity. However, in dopamine transporter knock-out mice that display a hyperactivity phenotype resulting from a persistent hyperdopaminergic state, these drugs antagonize hyperactivity. Under basal conditions, dopamine transporter knock-out mice show enhanced striatal DARPP-32 phosphorylation, activation of ERK, and inactivation of Akt as compared with wild-type littermates. However, administration of amphetamine or methylphenidate to these mice reveals that inhibition of ERK signaling is a common determinant for the ability of these drugs to antagonize hyperactivity. In contrast, psychostimulants activate ERK and induce hyperactivity in normal animals. In hyperactive mice psychostimulant-mediated behavioral inhibition and ERK regulation are also mimicked by the serotonergic drugs fluoxetine and 5-carboxamidotryptamine, thereby revealing the involvement of serotonin-dependent inhibition of striatal ERK signaling. Furthermore, direct inhibition of the ERK signaling cascade in vivo using the MEK inhibitor SL327 recapitulates the actions of psychostimulants in hyperactive mice and prevents the locomotor-enhancing effects of amphetamine in normal animals. These data suggest that the inhibitory action of psychostimulants on dopamine-dependent hyperactivity results from altered regulation of striatal ERK signaling. In addition, these results illustrate how altered homeostatic state of neurotransmission can influence in vivo signaling responses and biological actions of pharmacological agents used to manage psychiatric conditions such as Attention Deficit Hyperactivity Disorder (ADHD).
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PMID:Paradoxical striatal cellular signaling responses to psychostimulants in hyperactive mice. 1695 11

The primary mechanism for clearance of extracellular dopamine (DA) is uptake mediated by the dopamine transporter (DAT), which is governed, in part, by the number of functional DATs on the cell surface. Previous studies have shown that amphetamine (AMPH) decreases DAT cell surface expression, whereas insulin reverses this effect through the action of phosphatidylinositol 3-kinase (PI3K). Therefore, it is possible that AMPH causes DAT cell surface redistribution by inhibiting basal insulin signaling. Here, we show in a heterologous expression system and in murine striatal synaptosomes that AMPH causes a time-dependent decrease in the activity of Akt, a protein kinase immediately downstream of PI3K. This effect was blocked by the DAT inhibitor cocaine, suggesting that AMPH must interact with DAT to inhibit Akt. We also showed that AMPH is able to stimulate Ca2+/calmodulin-dependent kinase II (CaMKII) activity, both in the heterologous expression system as well as in murine striatal synaptosomes. The ability of AMPH to decrease Akt activity was blocked by the CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93), but not by its inactive analog 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN92). Furthermore, preincubation with KN93 prevented the AMPH-induced decrease in DAT cell surface expression. Thus, AMPH, but not cocaine, decreases Akt activity through a CaMKII-dependent pathway, thereby providing a novel mechanism by which AMPH regulates insulin signaling and DAT trafficking.
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PMID:Dopamine transporter activity mediates amphetamine-induced inhibition of Akt through a Ca2+/calmodulin-dependent kinase II-dependent mechanism. 1716 7

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor activated by a broad range of monoamines and amphetamine-related psychostimulants. Recent studies demonstrated wide distribution of TAAR1 in brain, coexpression of TAAR1 with dopamine transporter (DAT) in a subset of dopamine neurons in both mouse and rhesus monkey substantia nigra, and monoamine transporter-modulated activation. This study explored whether TAAR1 could influence DAT-mediated dopamine uptake and efflux. Rhesus monkey TAAR1 expressed with DAT in human embryonic kidney 293 cells was dose-dependently activated by dopamine or (+)-methamphetamine. This activation resulted in large cAMP increases and a transient reduction in [3H]dopamine accumulation within the cells, which was similar to the effect of dopamine D1 receptor (D1) or forskolin treatment. In addition, TAAR1 effects on dopamine uptake could be blocked by a protein kinase A or protein kinase C (PKC) inhibitor. [3H]Dopamine efflux assays performed in Dulbecco's modified Eagle's medium displayed a TAAR1-dependent spontaneous [3H]dopamine efflux that was dose-dependently augmented by dopamine or (+)-methamphetamine and that was blocked by either methylphenidate or a PKC inhibitor. DAT cells in Krebs-HEPES buffer had an apparent spontaneous [3H]dopamine loss, but it could not be blocked by either methylphenidate or a PKC inhibitor. Taken together, this study provides evidence that TAAR1 is involved in functional regulation of DAT and suggests that TAAR1 is a potentially important target for therapeutics for methamphetamine addiction.
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PMID:Trace amine-associated receptor 1 is a modulator of the dopamine transporter. 1723 99

The glial cell line-derived neurotrophic factor (GDNF) exerts trophic actions on a number of cell types, including mesencephalic dopaminergic (mDA) neurons. Using rat mesencephalic primary cultures enriched in mDA neurons, we show that protracted GDNF stimulation increases their survival and neurite outgrowth. It modulates the expression of genes essential for DA function (tyrosine hydroxylase, TH and dopamine transporter, dat) and of DA high affinity uptake. To identify genes involved in GDNF signaling pathways, we have used DNA microarray on mDA cultures stimulated with GDNF for 3 h. Here we show that GDNF signaling sequentially activates the genes encoding for the transcription factors EGR1 and TIEG. In addition, it increases the expression of cav1, which encodes for the major component of caveolae. GDNF also modulates the expression of the genes encoding for the Calcineurin subunits ppp3R1 and ppp3CB, and inhibits calcium-calmodulin-dependent protein kinase II beta isoform (CaMKIIbeta) gene expression. These proteins are involved in neuronal differentiation and synaptic plasticity. Moreover, GDNF stimulation down regulates the expression of the glycogen synthase kinase 3beta (gsk3beta) gene, involved in neuronal apoptosis. Using inhibitors of specific intracellular signal transduction pathways we show that changes of egr1, tieg, cav1, CaMkIIbeta and gsk3beta genes expression are extracellular-signal regulated kinases 1/2 (ERK)-dependent, while the cAMP-dependent protein kinase (PKA) pathway influences the up-regulation of ppp3R1 and ppp3CB gene expression. These results demonstrate that GDNF stimulation results in the transcriptional modulation of genes involved in neuronal plasticity and survival and in mDA function, mediated in part by ERK and PKA signaling.
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PMID:GDNF signaling in embryonic midbrain neurons in vitro. 1757 20

Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA-binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p-glycogen synthase kinase 3beta (GSK3beta) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIalpha, vascular early response gene, GAP-43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3beta signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3beta, p-Akt, and beta-catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile-X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.
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PMID:Activity-dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain. 1901 79

Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with protein kinase C (PKC)- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues, we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCalpha, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCalpha-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses.
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PMID:Proline-directed phosphorylation of the dopamine transporter N-terminal domain. 1914 7

This study reveals a novel receptor mechanism for methamphetamine action in dopamine transporter (DAT) regulation. Trace amine-associated receptor 1 (TAAR1) is expressed in brain dopaminergic nuclei and is activated by methamphetamine in vitro. Here, we show that methamphetamine interaction with TAAR1 inhibits [(3)H]dopamine uptake, enhances or induces [(3)H]dopamine efflux, and triggers DAT internalization. In time course assays in which methamphetamine and [(3)H]dopamine were concurrently loaded into cells or synaptosomes or in pretreatment assays in which methamphetamine was washed away before [(3)H]dopamine loading, methamphetamine caused a distinct inhibition in [(3)H]dopamine uptake in TAAR1 + DAT-cotransfected cells and in wild-type mouse and rhesus monkey striatal synaptosomes. This distinct uptake inhibition was not observed in DAT-only transfected cells or in TAAR1 knockout mouse striatal synaptosomes. In [(3)H]dopamine efflux assays using the same cell and synaptosome preparations, methamphetamine enhanced [(3)H]dopamine efflux at a high loading concentration of [(3)H]dopamine (1 muM) or induced [(3)H]dopamine efflux at a low loading concentration of [(3)H]dopamine (10 nM) in a TAAR1-dependent manner. In DAT biotinylation assays using the same cell and synaptosome preparations, we observed that 1 muM methamphetamine induced DAT internalization in a TAAR1-dependent manner. All these TAAR1-mediated effects of methamphetamine were blocked by the protein kinase inhibitors H89 [N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline] and/or 2-{8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methylindol-3-yl)maleimide (Ro32-0432), suggesting that methamphetamine interaction with TAAR1 triggers cellular phosphorylation cascades and leads to the observed effects of methamphetamine on DAT. These findings demonstrate a mediatory role of TAAR1 in methamphetamine action in DAT regulation and implicate this receptor as a potential target of therapeutics drugs for methamphetamine addiction.
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PMID:A receptor mechanism for methamphetamine action in dopamine transporter regulation in brain. 1936 8

Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP32) at Thr75 residue, but preserved D(2) receptor (D(2)R) function. However, although we demonstrated that striatal D(1) receptor (D(1)R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D(1)Rs since haloperidol, by blocking the tonic inhibition of D(2)R, unmasked a normal activation of striatal adenosine A(2A) receptor-mediated cAMP/PKA/DARPP32 cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation of D(1)R/cAMP/PKA/DARPP32 signaling in response to increased striatal extracellular dopamine levels.
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PMID:Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine. 2072 Jan 11


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