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Angus C Nairn

Publications and source records attributed to Angus C Nairn.

At least 55 records · Page 3Linked to original sources
PubMed2004

Functional roles of nonconserved structural segments in CFTR's NH2-terminal nucleotide binding domain.

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282-293) identified two regions absent from structures of all other NBDs determined so far, a "regulatory insertion" (residues 404-435) and a "regulatory extension" (residues 639-670), both positioned to impede formation of the putative NBD1-NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1-414 with residues 433-1480, or residues 1-633 with 668-1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.

Amino Acid Sequence↗
PubMed2004

Cytoplasmic localization of calcium/calmodulin-dependent protein kinase I-alpha depends on a nuclear export signal in its regulatory domain.

Calcium/calmodulin-dependent protein kinase I-alpha (CaMKI-alpha) is a ubiquitous cytosolic enzyme that phosphorylates a number of nuclear proteins in vitro and has been implicated in transcriptional regulation. We report that cytoplasmic localization of CaMKI-alpha depends on CRM1-mediated nuclear export mediated through a Rev-like nuclear export signal in the CaMKI-alpha regulatory domain. Interaction of CaMKI-alpha with a CRM1 complex in vitro is enhanced by incubation with calcium/calmodulin. Translocation of CaMKI-alpha into the nucleus involves a conserved sequence located within the catalytic core. Mutation of this sequence partially blocks nuclear entry of an export-impaired mutant of CaMKI-alpha.

Active Transport, Cell Nucleus↗
PubMed2004

PKC-alpha regulates cardiac contractility and propensity toward heart failure.

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

Animals↗
PubMed2004

Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses.

Synaptojanin 1 is a polyphosphoinositide phosphatase concentrated in presynaptic nerve terminals, where it dephosphorylates a pool of phosphatidylinositol 4,5-bisphosphate implicated in synaptic vesicle recycling. Like other proteins with a role in endocytosis, synaptojanin 1 undergoes constitutive phosphorylation in resting synapses and stimulation-dependent dephosphorylation by calcineurin. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates synaptojanin 1 and regulates its function both in vitro and in intact synaptosomes. Cdk5 phosphorylation inhibited the inositol 5-phosphatase activity of synaptojanin 1, whereas dephosphorylation by calcineurin stimulated such activity. The activity of synaptojanin 1 was also stimulated by its interaction with endophilin 1, its major binding partner at the synapse. Notably, Cdk5 phosphorylated serine 1144, which is adjacent to the endophilin binding site. Mutation of serine 1144 to aspartic acid to mimic phosphorylation by Cdk5 inhibited the interaction of synaptojanin 1 with endophilin 1. These results suggest that Cdk5 and calcineurin may have an antagonistic role in the regulation of synaptojanin 1 recruitment and activity, and therefore in the regulation of phosphatidylinositol 4,5-bisphosphate turnover at synapses.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The role of DARPP-32 in the actions of drugs of abuse.

The dopamine- and cAMP-regulated phosphoprotein, M(r) 32 kDa (DARPP-32), plays a key role in dopaminoceptive neurons in the neostriatum (and likely in other brain regions) in signal transduction pathways regulated by a variety of neurotransmitters, neuromodulators, and neuropeptides. Phosphorylation at Thr34 by protein kinase A converts DARPP-32 into a potent inhibitor of the multifunctional serine/threonine protein phosphatase, PP-1. Phosphorylation at Thr75 by Cdk5 converts DARPP-32 into an inhibitor of protein kinase A. The state of phosphorylation of DARPP-32 at Thr34 also depends on the phosphorylation state of Ser102 and Ser137, which are phosphorylated by CK2 and CK1, respectively. By virtue of its regulation of its four phosphorylation sites by a large number of physiological and pharmacological stimuli, and through its ability to modulate the activity of PP-1 and protein kinase A, DARPP-32 plays a key role in integrating a variety of electrophysiological, transcriptional, and behavioral responses. This review focuses on the critical role that DARPP-32 plays in mediating the actions of a broad range of drugs of abuse.

Animals↗
PubMed2004

Recent advances in neuroproteomics and potential application to studies of drug addiction.

The rapidly growing field of proteomics seeks to track changes in protein expression function that underlie the growth and differentiation of individual cell types, both during normal development and during the onset and progression of disease. Recent years have seen great strides in mRNA expression analysis, and the development of new technologies for protein profiling. However, current methods are limited to analysis of the relative expression level of only a few hundred to perhaps 2000 proteins, well below the ability of DNA microarrays to potentially interrogate the mRNA expression of more than 25,000 genes. Proteomics faces a special challenge in studies of the nervous system, where cellular and sub-cellular architecture is among the most complex in the body. This article presents an overview of current proteomic profiling technologies, reviews the recent use of some of these approaches in studies of the nervous system, and discusses the potential application of neuroproteomics to studies of drug addiction.

Animals↗
PubMed2004

Restoration of protein synthesis in heart and skeletal muscle after withdrawal of alcohol.

BACKGROUND: The rate of protein synthesis is diminished after chronic alcohol consumption through changes in both mRNA translation initiation and elongation. It remains unknown how long adverse effects of alcohol on protein synthesis persist after withdrawal from ethanol. METHODS: We examined the effect of removal of alcohol from the diet of rats for 72 hr after chronic alcohol exposure (16 weeks) on rates of protein synthesis and potential mechanisms for controlling mRNA translation in heart, skeletal muscle, and liver. Rates of protein synthesis were measured after intravenous infusion of [3H]-l-phenylalanine. The formation of active eukaryotic initiation factor (eIF)4E.eIF4G complex, the cellular content of eukaryotic elongation factor (eEF)1A and eEF2, and the phosphorylation state of eEF2 and S6K1 were measured in each tissue. RESULTS: Withdrawal of alcohol from the diet restored protein synthesis in heart and skeletal muscle to values obtained in pair-fed control rats not exposed to alcohol. However, the organ weight and protein content per muscle was not affected by withdrawal of alcohol from the diet. In both heart and skeletal muscle, the restoration of protein synthesis correlated with reversal of defects in the formation of active eIF4E.eIF4G complex and eEF1A content. Myocardial eEF2 content was also restored to control values after withdrawal of alcohol from the diet. In the gastrocnemius, there was a decrease in the cellular content of eEF2. The lower eIF2 content may have been counterbalanced by an increased activity of eEF2 through a reduction in the phosphorylation state of eEF2 allowing protein synthesis to proceed unimpeded. CONCLUSIONS: These studies indicate that changes in protein metabolism observed during chronic alcohol intake are reversible and do not, at this stage, represent an irreversible change in cardiac or skeletal muscle.

Animals↗
PubMed2004

Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation.

The regulation of adenosine kinase (AK) activity has the potential to control intracellular and interstitial adenosine (Ado) concentrations. In an effort to study the role of AK in Ado homeostasis in the central nervous system, two isoforms of the enzyme were cloned from a mouse brain cDNA library. Following overexpression in bacterial cells, the corresponding proteins were purified to homogeneity. Both isoforms were enzymatically active and found to possess K(m) and V(max) values in agreement with kinetic parameters described for other forms of AK. The distribution of AK in discrete brain regions and various peripheral tissues was defined. To investigate the possibility that AK activity is regulated by protein phosphorylation, a panel of protein kinases was screened for ability to phosphorylate recombinant mouse AK. Data from these in vitro phosphorylation studies suggest that AK is most likely not an efficient substrate for PKA, PKG, CaMKII, CK1, CK2, MAPK, Cdk1, or Cdk5. PKC was found to phosphorylate recombinant AK efficiently in vitro. Further analysis revealed, however, that this PKC-dependent phosphorylation occurred at one or more serine residues associated with the N-terminal affinity tag used for protein purification.

Adenosine↗
PubMed2004

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin.

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.

Actin Cytoskeleton↗
PubMed2004

Differential regulation of dopamine D1 and D2 signaling by nicotine in neostriatal neurons.

Nicotine, acting on nicotinic acetylcholine receptors (nAChRs) expressed at pre-synaptic dopaminergic terminals, has been shown to stimulate the release of dopamine in the neostriatum. However, the molecular consequences of pre-synaptic nAChR activation in post-synaptic neostriatal neurons are not clearly understood. Here, we investigated the effect of nAChR activation on dopaminergic signaling in medium spiny neurons by measuring phosphorylated DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) at Thr34 (the PKA-site) in mouse neostriatal slices. Nicotine produced dose-dependent responses, with a low concentration (1 microm) causing a sustained decrease in DARPP-32 Thr34 phosphorylation and a high concentration (100 microm) causing a transient increase in DARPP-32 Thr34 phosphorylation. Depending on the concentration of nicotine, either dopamine D2 or D1 receptor signaling was predominantly activated. Nicotine at a low concentration (1 microm) activated dopamine D2 receptor signaling in striatopallidal/indirect pathway neurons, likely by activating alpha4beta2* nAChRs at dopaminergic terminals. Nicotine at a high concentration (100 microm) activated dopamine D1 receptor signaling in striatonigral/direct pathway neurons, likely by activating (i) alpha4beta2* nAChRs at dopaminergic terminals and (ii) alpha7 nAChRs at glutamatergic terminals, which, by stimulating the release of glutamate, activated NMDA/AMPA receptors at dopaminergic terminals. The differential effects of low and high nicotine concentrations on D2- and D1-dependent signaling pathways in striatal neurons may contribute to dose-dependent actions of this drug of abuse.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2004

DARPP-32: an integrator of neurotransmission.

Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32), was identified initially as a major target for dopamine and protein kinase A (PKA) in striatum. However, recent advances now indicate that regulation of the state of DARPP-32 phosphorylation provides a mechanism for integrating information arriving at dopaminoceptive neurons, in multiple brain regions, via a variety of neurotransmitters, neuromodulators, neuropeptides, and steroid hormones. Activation of PKA or PKG stimulates DARPP-32 phosphorylation at Thr34 and thereby converts DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP-1). DARPP-32 is also phosphorylated at Thr75 by Cdk5 and this converts DARPP-32 into an inhibitor of PKA. Thus, DARPP-32 has the unique property of being a dual-function protein, acting either as an inhibitor of PP-1 or of PKA. The state of phosphorylation of DARPP-32 at Thr34 depends on the phosphorylation state of two serine residues, Ser102 and Ser137, which are phosphorylated by CK2 and CK1, respectively. By virtue of its ability to modulate the activity of PP-1 and PKA, DARPP-32 is critically involved in regulating electrophysiological, transcriptional, and behavioral responses to physiological and pharmacological stimuli, including antidepressants, neuroleptics, and drugs of abuse.

Animals↗
PubMed2003

Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3.

Cell surface receptor membrane localization is strongly dependent on protein-protein interactions often involving regulation by phosphorylation/dephosphorylation of the intracellular domains of membrane proteins. The present study was carried out to identify metabotropic glutamate receptor (mGluR) 3 regulatory binding proteins. Using the yeast two-hybrid technique, we found that the 50-aa C-terminal cytoplasmic tail of mGluR3 interacts specifically with protein phosphatase 2Calpha (PP2Calpha). This interaction was confirmed by GST pull-down and coimmunoprecipitation assays. mGluR3 interacts with PP2Calpha, beta, gamma, and delta isoforms; however, among the mGluR family only mGluR3 interacted with PP2C. The minimal interacting domain of mGluR3 comprised residues 836-855. Alignment between mGluR3 and mGluR2, a closely related group II receptor, indicated that this domain is not conserved between the two receptors. The mGluR3 cytoplasmic C-terminal tail contains one phosphorylation site for protein kinase A (Ser-845), but the phosphatase that dephosphorylates this site has not been previously identified. We find that PP2C, but not PP1, PP2A, or PP2B, dephosphorylates the mGluR3 cytoplasmic tail in vitro. The dephosphorylated form of the mGluR3 cytoplasmic tail, but not the equivalent region of mGluR2, inhibited PP2C assayed by using [32P]casein as a substrate. However, phosphorylation of the mGluR3 cytoplasmic tail at Ser-845 inhibits the interaction with PP2C. These results indicate distinct functions for mGluR2 and mGluR3 and suggest a dynamic regulation of mGluR3 by PP2C.

Amino Acid Sequence↗
PubMed2003

The selective inhibition of phosphatases by natural toxins: the anhydride domain of tautomycin is not a primary factor in controlling PP1/PP2A selectivity.

Analogues of the potent and moderately selective PP1/PP2A inhibitor tautomycin (TM) were prepared with modifications in the C1'-C7' anhydride moiety. While all retain varying degrees of activity within a 3000-fold range of potencies, they also show remarkable constancy in their IC(50) ratios, suggesting that the anhydride moiety is not critical in controlling the selectivity of inhibition.

Anhydrides↗
PubMed2003

A new model of the tautomycin-PP1 complex that is not analogous to the corresponding okadaic acid structure.

A revised model of PP1-tautomycin (TM) complex suggests that this toxin does not bind in a conformation analogous to its structural cousin okadaic acid (OA), as has been assumed, but instead more resembles the mode of binding adopted by calyculin. This model rationalizes the unexpected potency of a truncated TM analogue lacking the bicyclic ketal common to TM and OA.

Enzyme Inhibitors↗
PubMed2003

Glucose regulates EF-2 phosphorylation and protein translation by a protein phosphatase-2A-dependent mechanism in INS-1-derived 832/13 cells.

The role of elongation factor (EF)-2 phosphorylation in the regulation of pancreatic beta-cell protein synthesis by glucose was investigated in the INS-1-derived cell line 832/13. Incubation of cells in media containing 1 mm glucose resulted in a progressive increase in EF-2 phosphorylation that was maximal by 1-2 h. Readdition of 10 mm glucose promoted a rapid dephosphorylation of EF-2 that was complete in 10 min and maintained over the ensuing 2 h. Similar results were obtained using primary rat islets or Min-6 insulinoma cells. The glucose effect in 832/13 cells was replicated by addition of pyruvate or alpha-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nm insulin, 30 mm potassium chloride, or 0.25 mm diazoxide, indicating that insulin secretion and/or depolarization of beta cells was not required. The locus of the high glucose effect appeared to be protein phosphatase-2A, the principal phosphatase acting on EF-2. Protein phosphatase-2A activity was stimulated by glucose addition to 832/13 cells, but neither protein phosphatase-1 nor calmodulin kinase III (EF-2 kinase) activity was affected under these conditions. The slower rephosphorylation of EF-2 during the transition from high to low glucose may involve effects on EF-2 kinase activity. Addition of 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside in high glucose led to a marked stimulation of EF-2 phosphorylation, consistent with the possibility that increased AMP kinase activity in low glucose stimulates EF-2 kinase. In parallel with the effects on EF-2 dephosphorylation, addition of high glucose to 832/13 cells markedly increased the incorporation of [(35)S]methionine into total protein. Taken together, these results suggest that modulation of extracellular glucose impacts protein translation rate in beta cells at least in part through regulation of the elongation step, via phosphorylation/dephosphorylation of EF-2.

Adenylate Kinase↗
PubMed2003

PNUTS, a protein phosphatase 1 (PP1) nuclear targeting subunit. Characterization of its PP1- and RNA-binding domains and regulation by phosphorylation.

PNUTS, Phosphatase 1 NUclear Targeting Subunit, is a recently described protein that targets protein phosphatase 1 (PP1) to the nucleus. In the present study, we characterized the biochemical properties of PNUTS. A variety of truncation and site-directed mutants of PNUTS was prepared and expressed either as glutathione S-transferase fusion proteins in Escherichia coli or as FLAG-tagged proteins in 293T cells. A 50-amino acid domain in the center of PNUTS mediated both high affinity PP1 binding and inhibition of PP1 activity. The PP1-binding domain is related to a motif found in several other PP1-binding proteins but is distinct in that Trp replaces Phe. Mutation of the Trp residue essentially abolished the ability of PNUTS to bind to and inhibit PP1. The central PP1-binding domain of PNUTS was an effective substrate for protein kinase A in vitro, and phosphorylation substantially reduced the ability of PNUTS to bind to PP1 in vitro and following stimulation of protein kinase A in intact cells. In vitro RNA binding experiments showed that a C-terminal region including several RGG motifs and a novel repeat domain rich in His and Gly interacted with mRNA and single-stranded DNA. PNUTS exhibited selective binding for poly(A) and poly(G) compared with poly(U) or poly(C) ribonucleotide homopolymers, with specificity being mediated by distinct regions within the domain rich in His and Gly and the domain containing the RGG motifs. Finally, a PNUTS-PP1 complex was isolated from mammalian cell lysates using RNA-conjugated beads. Together, these studies support a role for PNUTS in protein kinase A-regulated targeting of PP1 to specific RNA-associated complexes in the nucleus.

Amino Acid Motifs↗
PubMed2003

Metabotropic mGlu5 receptors regulate adenosine A2A receptor signaling.

Dopamine, by activating dopamine D1-type receptors, and adenosine, by activating adenosine A(2A) receptors, stimulate phosphorylation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000) at Thr-34. In this study, we investigated the effect of metabotropic glutamate (mGlu) receptors on DARPP-32 phosphorylation at Thr-34 in neostriatal slices. A broad-spectrum mGlu receptor agonist, trans-ACPD, and a group I mGlu receptor agonist, DHPG, stimulated DARPP-32 phosphorylation at Thr-34. Studies with mGlu receptor antagonists revealed that the effects of trans-ACPD and DHPG were mediated through activation of mGlu5 receptors. The action of mGlu5 receptors required activation of adenosine A(2A) receptors by endogenous adenosine. Conversely, the action of adenosine A(2A) receptors required activation of mGlu5 receptors by endogenous glutamate. Coactivation of mGlu5 and adenosine A(2A) receptors by exogenous agonists synergistically increased DARPP-32 phosphorylation. mGlu5 receptors did not require activation of dopamine D1-type receptors by endogenous dopamine, nor did dopamine D1-type receptors require activation of mGlu5 receptors by endogenous glutamate. DHPG potentiated the effect of forskolin, but not that of 8-bromo-cAMP, and stimulated DARPP-32 phosphorylation in the presence of the phosphodiesterase inhibitor IBMX, suggesting that mGlu5 receptors stimulate the rate of cAMP formation coupled to adenosine A(2A) receptors. The action of mGlu5 receptors was attenuated by inhibitors of extracellular signal-regulated kinase, but not by inhibitors of phospholipase C, p38, casein kinase 1, or Cdk5. The results demonstrate that mGlu5 receptors potentiate adenosine A(2A)DARPP-32 signaling by stimulating the adenosine A(2A) receptor-mediated formation of cAMP in an extracellular signal-regulated kinase-dependent manner.

1-Methyl-3-isobutylxanthine↗