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Angus C Nairn

Publications and source records attributed to Angus C Nairn.

At least 73 records · Page 4Linked to original sources
PubMed2003

Regulation of AMPA receptor dephosphorylation by glutamate receptor agonists.

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.

Animals↗
PubMed2003

Thr123 of rat G-substrate contributes to its action as a protein phosphatase inhibitor.

Rat G-substrate cDNA was isolated from a cerebellar library and characterized. The deduced amino acid sequence of rat G-substrate contained two putative phosphorylation sites for PKG at Thr72 and Thr123; the amino acid sequences (KPRRKDT(p)PA) around these sites are conserved in human, mouse and rabbit. G-substrate phosphorylated by PKG inhibited the catalytic subunits of both protein phosphatase-1 (IC(50) 14.1 nM) and -2A (IC(50) 5.9 nM). Mutation of Thr123 (site 2) to Ala significantly reduced the inhibition of both PP-1 and PP-2A, while mutation of Thr72 (site 1) to Ala had little effect on inhibitory activity. In situ hybridization analysis revealed that G-substrate mRNA was localized exclusively in cerebellar Purkinje cells. Immunoperoxidase staining showed that in Purkinje cells, G-substrate was present in somata, dendrites and axons. In rat cerebellar slices, activation of PKG with a nitric oxide (NO) donor, NOR3, or 8-Br-cGMP, increased phosphorylation of G-substrate, as demonstrated with a phosphorylation-specific antibody. These results characterize further the inhibition of PP-1 and PP-2A by phospho-G-substrate, and demonstrate its physiological phosphorylation in rat Purkinje cells.

Amino Acid Sequence↗
PubMed2003

Crystal structure of a tetradecameric assembly of the association domain of Ca2+/calmodulin-dependent kinase II.

We report the crystal structure of the 143 residue association domain of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The association domain forms a hub-like assembly, composed of two rings of seven protomers each, which are stacked head to head and held together by extensive interfaces. The tetradecameric organization of the assembly was confirmed by analytical ultracentrifugation and multiangle light scattering. Individual protomers form wedge-shaped structures from which N-terminal helical segments that connect to the kinase domain extend toward the equatorial plane of the assembly, consistent with the arrangement of the kinase domains in a second outer ring. A deep and highly conserved pocket present within the association domain may serve as a docking site for proteins that interact with CaMKII.

Amino Acid Sequence↗
PubMed2003

NMDA-mediated activation of the tyrosine phosphatase STEP regulates the duration of ERK signaling.

The intracellular mechanism(s) by which a cell determines the duration of extracellular signal-regulated kinase (ERK) activation is not well understood. We have investigated the role of STEP, a striatal-enriched tyrosine phosphatase, in the regulation of ERK activity in rat neurons. Glutamate-mediated activation of NMDA receptors leads to the rapid but transient phosphorylation of ERK in cultured neurons. Here we show that activation of NMDA receptors led to activation of STEP, which limited the duration of ERK activity as well as its translocation to the nucleus and its subsequent downstream nuclear signaling. In neurons, STEP is phosphorylated and inactive under basal conditions. NMDA-mediated influx of Ca(2+), but not increased intracellular Ca(2+) from other sources, leads to activation of the Ca(2+)-dependent phosphatase calcineurin and the dephosphorylation and activation of STEP. We have identified an important mechanism involved in the regulation of ERK activity in neurons that highlights the complex interplay between serine/threonine and tyrosine kinases and phosphatases.

Active Transport, Cell Nucleus↗
PubMed2003

Regulation of ania-6 splice variants by distinct signaling pathways in striatal neurons.

The striatum is a brain region involved in motor control and in diverse forms of implicit memory. It is also involved in the pathogenesis of many significant human disorders, including drug addiction, that are thought to involve adaptive changes in gene expression. We have previously shown that the cyclin L, ania-6, is expressed as at least two splice forms, which are differentially regulated in striatal neurons by different neurotransmitters. Here, we report that ania-6 transcription is mostly regulated via cAMP response element binding protein (CREB), but that signaling pathways that converge on CREB at the transcriptional level produce different effects on splicing and neuronal gene expression. Glutamate induced a long ania-6 mRNA that encodes a truncated form of the cyclin. This effect depended on the activation of NMDA receptors but was independent of both calcium/calmodulin-dependent protein kinases (CaMK) and extracellular regulated kinase (ERK). Forskolin or brain-derived neurotropic factor (BDNF) induced a short ania-6 mRNA, that encodes the full-length cyclin, and this induction depended on ERK. However, KCl-mediated induction of ania-6 short mRNA, which required activation of L-type calcium channels, was independent of ERK but depended on CaMK. These data suggest that different neuronal signals can differentially regulate splicing and that different intracellular pathways can be recruited to yield a given splice variant.

Alternative Splicing↗
PubMed2003

Effect of methylphenidate on dopamine/DARPP signalling in adult, but not young, mice.

Methylphenidate (MPH), a dopamine uptake inhibitor, is the most commonly prescribed drug for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children. We examined the effect of MPH on dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) phosphorylation at Thr34 (PKA-site) and Thr75 (Cdk5-site) using neostriatal slices from young (14-15- and 21-22-day-old) and adult (6-8-week-old) mice. MPH increased DARPP-32 Thr34 phosphorylation and decreased Thr75 phosphorylation in slices from adult mice. The effect of MPH was blocked by a dopamine D1 antagonist, SCH23390. In slices from young mice, MPH did not affect DARPP-32 phosphorylation. As with MPH, cocaine stimulated DARPP-32 Thr34 phosphorylation in slices from adult, but not from young mice. In contrast, a dopamine D1 agonist, SKF81297, regulated DARPP-32 phosphorylation comparably in slices from young and adult mice, as did methamphetamine, a dopamine releaser. The results suggest that dopamine synthesis and the dopamine transporter are functional at dopaminergic terminals in young mice. In contrast, the lack of effect of MPH in young mice is likely attributable to immature development of the machinery that regulates vesicular dopamine release.

Aging↗
PubMed2003

On the mechanism of MgATP-dependent gating of CFTR Cl- channels.

CFTR, the product of the gene mutated in cystic fibrosis, is an ATPase that functions as a Cl(-) channel in which bursts of openings separate relatively long interburst closed times (tauib). Channel gating is controlled by phosphorylation and MgATP, but the underlying molecular mechanisms remain controversial. To investigate them, we expressed CFTR channels in Xenopus oocytes and examined, in excised patches, how gating kinetics of phosphorylated channels were affected by changes in [MgATP], by alterations in the chemical structure of the activating nucleotide, and by mutations expected to impair nucleotide hydrolysis and/or diminish nucleotide binding affinity. The rate of opening to a burst (1/tauib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR's nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Burst duration of neither wild-type nor mutant channels was much influenced by [MgATP]. Poorly hydrolyzable nucleotide analogs, MgAMPPNP, MgAMPPCP, and MgATPgammaS, could open CFTR channels, but only to a maximal rate of opening approximately 20-fold lower than attained by MgATP acting on the same channels. NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Corresponding NBD1 mutations did not affect timing of burst termination in normal, hydrolytic conditions. However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. In light of recent biochemical and structural data, the results suggest that: nucleotide binding to both NBDs precedes channel opening; at saturating nucleotide concentrations the rate of opening to a burst is influenced by the structure of the phosphate chain of the activating nucleotide; normal, rapid exit from bursts occurs after hydrolysis of the nucleotide at NBD2, without requiring a further nucleotide binding step; if hydrolysis at NBD2 is prevented, exit from bursts occurs through a slower pathway, the rate of which is modulated by the structure of the NBD1 catalytic site and its bound nucleotide. Based on these and other results, we propose a mechanism linking hydrolytic and gating cycles via ATP-driven dimerization of CFTR's NBDs.

Adenosine Triphosphate↗
PubMed2003

Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.

CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.

Adenosine Diphosphate↗
PubMed2002

Phosphorylation of spinophilin modulates its interaction with actin filaments.

Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission.

Actins↗
PubMed2002

Nerve growth factor controls GAP-43 mRNA stability via the phosphoprotein ARPP-19.

The membrane phosphoprotein GAP-43 is involved in axon growth and synaptic plasticity. In PC12 pheochromocytoma cells, induction of a neuronal phenotype by nerve growth factor (NGF) is accompanied by a marked increase in GAP-43 levels. NGF regulates GAP-43 expression by altering the half-life of its mRNA. We report here that the phosphoprotein ARPP-19 mediates this regulation. In an NGF-dependent manner, ARPP-19 bound to a region in the 3' end of GAP-43 mRNA previously found to be important for regulating the half-life of the mRNA. Overexpression of wild-type ARPP-19 in PC12 cells increased the NGF-dependent expression of a reporter construct linked to the critical 3' region of GAP-43 mRNA. Mutation of serine 104, the site of phosphorylation by protein kinase A in ARPP-19, to either alanine or aspartate abolished this regulation in PC12 cells. These findings demonstrate that ARPP-19 is an important link between NGF signaling and post-transcriptional control of neuronal gene expression.

Animals↗
PubMed2002

Mechanism of regulation of casein kinase I activity by group I metabotropic glutamate receptors.

Previously, we reported that (S)-3,5-dihydroxypenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRs), stimulates CK1 and Cdk5 kinase activities in neostriatal neurons, leading to enhanced phosphorylation, respectively, of Ser-137 and Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, 32 kDa). We have now investigated the signaling pathway that leads from mGluRs to casein kinase 1 (CK1) activation. In mouse neostriatal slices, the effect of DHPG on phosphorylation of Ser-137 or Thr-75 of DARPP-32 was blocked by the phospholipase Cbeta inhibitor, the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), and the calcineurin inhibitor cyclosporin A. In neuroblastoma N2a cells, the effect of DHPG on the activity of transfected HA-tagged CK1(epsilon) was blocked by BAPTA/AM and cyclosporin A. In neostriatal slices, the effect of DHPG on Cdk5 activity was also abolished by BAPTA/AM and cyclosporin A, presumably through blocking activation of CK1. Metabolic labeling studies and phosphopeptide mapping revealed that a set of C-terminal sites in HA-CK1epsilon were transiently dephosphorylated in N2a cells upon treatment with DHPG, and this was blocked by cyclosporin A. A mutant CK1epsilon with a nonphosphorylatable C-terminal domain was not activated by DHPG. Together, these studies suggest that DHPG activates CK1(epsilon) via Ca(2+)-dependent stimulation of calcineurin and subsequent dephosphorylation of inhibitory C-terminal autophosphorylation sites.

Animals↗
PubMed2002

Involvement of DARPP-32 phosphorylation in the stimulant action of caffeine.

Caffeine has been imbibed since ancient times in tea and coffee, and more recently in colas. Caffeine owes its psychostimulant action to a blockade of adenosine A(2A) receptors, but little is known about its intracellular mechanism of action. Here we show that the stimulatory effect of caffeine on motor activity in mice was greatly reduced following genetic deletion of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of relative molecular mass 32,000). Results virtually identical to those seen with caffeine were obtained with the selective A(2A) antagonist SCH 58261. The depressant effect of the A(2A) receptor agonist, CGS 21680, on motor activity was also greatly attenuated in DARPP-32 knockout mice. In support of a role for DARPP-32 in the action of caffeine, we found that, in striata of intact mice, caffeine increased the state of phosphorylation of DARPP-32 at Thr 75. Caffeine increased Thr 75 phosphorylation through inhibition of PP-2A-catalysed dephosphorylation, rather than through stimulation of cyclin-dependent kinase 5 (Cdk5)-catalysed phosphorylation, of this residue. Together, these studies demonstrate the involvement of DARPP-32 and its phosphorylation/dephosphorylation in the stimulant action of caffeine.

Adenosine↗
PubMed2002

A direct test of the reductionist approach to structural studies of calmodulin activity: relevance of peptide models of target proteins.

Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM. CaMKIp complex indicate large differences throughout the CaM sequence. Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme. NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain. Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM. CaMKI complexes. Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM. In this context, the observed similarity between CaM. CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme.

Animals↗
PubMed2002

The carboxyl-terminus of BACE contains a sorting signal that regulates BACE trafficking but not the formation of total A(beta).

BACE (beta-site APP cleaving enzyme) has been recently proposed as the major aspartyl protease displaying beta secretase activity in neurons. The C-terminal domain of BACE contains a dileucine motif (LL499/500) that can potentially regulate its trafficking and endocytosis, and an adjacent serine, which is a potential phosphorylation site (S498) that could modulate the activity of the LL motif. In this paper we show that S498 is phosphorylated by casein kinase 1 (CKI). Mutating the LL to dialanine (AA) caused an increase in the levels of mature BACE. The LL to AA mutation increased levels of BACE on the cell surface and decreased the internalization of BACE. Mutating the S498 to alanine did not alter levels of cell surface BACE. Mutating either the leucines or the serine did not alter the secretion of A(beta). Our data are consistent with a role for the cytoplasmic domain in regulating BACE trafficking and localization.

Amino Acid Sequence↗
PubMed2002

Zinc-induced inhibition of protein synthesis and reduction of connexin-43 expression and intercellular communication in mouse cortical astrocytes.

Zinc released from a subpopulation of glutamatergic synapses, mainly localized in the cerebral cortex and the hippocampus, facilitates or reduces glutamatergic transmission by acting on neuronal AMPA and NMDA receptors, respectively. However, neurons are not the only targets of zinc. In the present study, we provide evidence that zinc inhibits protein synthesis in cultured astrocytes from the cerebral cortex of embryonic mice. This inhibition, which reached 85% in the presence of 100 micro m zinc, was partially and slowly reversible and resulted from the successive inhibition of the elongation and the initiation steps of the protein translation process. This was assessed by measuring the phosphorylation level of the elongation factor eEF-2 and of the alpha subunit of the initiation factor eIF-2. Due to the rapid turnover of connexin-43 that forms junction channels in cultured astrocytes, the zinc-induced decrease of protein synthesis led to a partial disappearance of connexin-43, which was associated with an inhibition of the cellular coupling in the astrocytic syncitium. In conclusion, zinc not only inhibits protein synthesis in neurons, as previously demonstrated, but also in astrocytes. The resulting decrease in the intercellular communication between astrocytes should alter the function of surrounding neurons as well as their survival.

Animals↗
PubMed2002

Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A.

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M (r) 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca(2+) -dependent activation of the Ca(2+) -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca(2+) -dependent activation of dephosphorylation by protein phosphatase-2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP-32 by different protein phosphatases.

Animals↗
PubMed2002

Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel.

The roles played by ATP binding and hydrolysis in the complex mechanisms that open and close cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels remain controversial. In this work, the contributions made by ATP and Mg(2+) ions to the gating of phosphorylated cardiac CFTR channels were evaluated separately by measuring the rates of opening and closing of single channels in excised patches exposed to solutions in which [ATP] and [Mg(2+)] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg(2+)-dependent step that followed binding of both ATP and Mg(2+). Once a channel had opened, sudden withdrawal of all Mg(2+) and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg(2+) ions alone could close it, and the closing rate increased with [Mg(2+)] over the micromolar range (half maximal at approximately 50 microM [Mg(2+)]). A simple interpretation is that channel closing is stoichiometrically coupled to hydrolysis of an ATP molecule that remains tightly associated with the open CFTR channel despite continuous washing. If correct, that ATP molecule appears able to reside for over a minute in the catalytic site that controls channel closing, implying that the site must entrap, or have an intrinsically high apparent affinity for, ATP, even without a Mg(2+) ion. Such stabilization of the open-channel conformation of CFTR by tight binding, or occlusion, of an ATP molecule echoes the stabilization of the active conformation of a G protein by GTP.

Adenosine Triphosphate↗