Insulin trigger, cyclic AMP-dependent activation and phosphorylation of a plasma membrane cyclic AMP phosphodiesterase

Regulation of blood glucose levels by the liver is primarily achieved by the action of two peptide hormones, insulin and glucagon, which bind to specific receptors associated with the hepatocyte plasma membrane. Whilst the molecular action of glucagon at the level of the cell plasma membrane in activating adenylate cyclase is relatively well understood, we know little, if anything, of the molecular consequences of insulin occupying its receptor. We demonstrate here that insulin, at physiologically relevant concentrations, can trigger the cyclic AMP-dependent activation and phosphorylation of a low Km cyclic AMP phosphodiesterase attached to the liver plasma membrane. Such an effect may in part explain the ability of insulin to inhibit the increase in cellular cyclic AMP content that glucagon alone produces by activation of adenylate cyclase. Our observation that basal, intracellular cyclic AMP levels are insufficient to allow insulin to activate the cyclic AMP phosphodiesterase, yet those cyclic AMP levels achieved after exposure of the cells to glucagon are sufficient, gives a molecular rationale to Butcher and Sutherland's proposal that it is necessary to first elevate cellular cyclic AMP levels before they can be depressed by insulin.     

Photochemical attachment of cyclic AMP binding protein(s) to the nuclear genome.

The nature of the cyclic AMP-receptor-nucleus interactions was examined by a novel combination of two photoreactions. A photosensitive derivative of cyclic AMP, N6-butyryl cyclic AMP, was covalently attached to its cytoplasmic receptod by photo-affinity labelling and this receptor complex was photo-crosslinked by the DNA in the rat liver nuclei. The photolytic reactions seemed to be specific since stable links were formed only when substantial noncovalent binding occured.     

Structural basis for AMP binding to mammalian AMP-activated protein kinase

AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type II diabetes treatment. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways. We report here the crystal structure of the regulatory fragment of mammalian AMPK in complexes with AMP and ATP. The phosphate groups of AMP/ATP lie in a groove on the surface of the gamma domain, which is lined with basic residues, many of which are associated with disease-causing mutations. Structural and solution studies reveal that two sites on the gamma domain bind either AMP or Mg.ATP, whereas a third site contains a tightly bound AMP that does not exchange. Our binding studies indicate that under physiological conditions AMPK mainly exists in its inactive form in complex with Mg.ATP, which is much more abundant than AMP. Our modelling studies suggest how changes in the concentration of AMP ([AMP]) enhance AMPK activity levels. The structure also suggests a mechanism for propagating AMP/ATP signalling whereby a phosphorylated residue from the alpha and/or beta subunits binds to the gamma subunit in the presence of AMP but not when ATP is bound.     

Structure and conserved RNA binding of the PAZ domain

The discovery of RNA-mediated gene-silencing pathways, including RNA interference, highlights a fundamental role of short RNAs in eukaryotic gene regulation and antiviral defence. Members of the Dicer and Argonaute protein families are essential components of these RNA-silencing pathways. Notably, these two families possess an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose biochemical function is unknown. Here we report the nuclear magnetic resonance solution structure of the PAZ domain from Drosophila melanogaster Argonaute 1 (Ago1). The structure consists of a left-handed, six-stranded beta-barrel capped at one end by two alpha-helices and wrapped on one side by a distinctive appendage, which comprises a long beta-hairpin and a short alpha-helix. Using structural and biochemical analyses, we demonstrate that the PAZ domain binds a 5-nucleotide RNA with 1:1 stoichiometry. We map the RNA-binding surface to the open face of the beta-barrel, which contains amino acids conserved within the PAZ domain family, and we define the 5'-to-3' orientation of single-stranded RNA bound within that site. Furthermore, we show that PAZ domains from different human Argonaute proteins also bind RNA, establishing a conserved function for this domain.     

Modulation of the two promoters of the galactose operon of Escherichia coli.

The gal operon of Escherichia coli is controlled by two independent promotors--one is activated and the other inhibited by cyclic AMP and cyclic AMP receptor protein. The two promotors are modulated, however, by the same operator locus and receptor protein.     

Correction of cell-cell communication defect by introduction of a protein kinase into mutant cells

The cell-to-cell permeability of the junctions of various cultured mammalian cell types depends on the concentration of intracellular cyclic AMP [( cAMP]i). The permeability rises when the cells are supplied with exogenous cyclic AMP or when their cyclic AMP synthesis is stimulated with choleragen or hormones; it falls when [cAMP]i is lowered by application of serum or due to increase in cell density. The rise and fall in permeability take several hours to develop (the rise is protein synthesis-dependent) and they occur concurrently with the rise and fall in the number of intramembrane particles of the gap junctions, which probably embody the cell-to-cell channels. Is this permeability regulation mediated by phosphorylating protein kinase? In many eukaryotes, the cyclic AMP receptor is a protein kinase consisting of a pair of regulatory subunits and a pair of catalytic subunits. The latter dissociate from the holoenzyme as the cyclic AMP binds to the regulatory subunits and, in this dissociated form, catalyse the phosphorylation of the target. The regulatory subunit occurs in two isoenzyme forms, I and II. The catalytic subunit seems invariant; subunits from different isoenzymes can substitute for each other. We show here that a mutant cell lacking the isoenzyme I is deficient in permeable junctions, and that this junctional defect is corrected when the mutant is supplied with exogenous catalytic subunit.     

Patch- and voltage-clamp analysis of cyclic AMP-stimulated inward current underlying neurone bursting

The second messenger cyclic AMP has been variously reported to affect the electrical activity of different neurones by decreasing outward potassium current, increasing outward current and increasing inward current. The recently developed patch clamp method of recording single ionic channels allows direct measurement of the action of cyclic AMP on membrane conductances. Using the patch clamp, the closure of potassium channels by cyclic AMP has previously been documented on the single channel level. We report here that in a bursting molluscan neurone, intracellular iontophoresis of cyclic AMP under voltage clamp elicits an inward current of maximal amplitude in the pacemaker voltage region. Patch-clamp analysis reveals inward channels whose opening frequency is augmented by cyclic AMP stimulation and whose activity accompanies burst episodes. Channel opening frequency is significantly increased by depolarization of the whole soma, but not by focal depolarization of the patch; this may reflect the action of another second messenger that acts in concert with cyclic AMP to confer voltage sensitivity.     

Protein activity regulation by conformational entropy

How the interplay between protein structure and internal dynamics regulates protein function is poorly understood. Often, ligand binding, post-translational modifications and mutations modify protein activity in a manner that is not possible to rationalize solely on the basis of structural data. It is likely that changes in the internal motions of proteins have a major role in regulating protein activity, but the nature of their contributions remains elusive, especially in quantitative terms. Here we show that changes in conformational entropy can determine whether protein-ligand interactions will occur, even among protein complexes with identical binding interfaces. We have used NMR spectroscopy to determine the changes in structure and internal dynamics that are elicited by the binding of DNA to several variants of the catabolite activator protein (CAP) that differentially populate the inactive and active DNA-binding domain states. We found that the CAP variants have markedly different affinities for DNA, despite the CAP?DNA-binding interfaces being essentially identical in the various complexes. Combined with thermodynamic data, the results show that conformational entropy changes can inhibit the binding of CAP variants that are structurally poised for optimal DNA binding or can stimulate the binding activity of CAP variants that only transiently populate the DNA-binding-domain active state. Collectively, the data show how changes in fast internal dynamics (conformational entropy) and slow internal dynamics (energetically excited conformational states) can regulate binding activity in a way that cannot be predicted on the basis of the protein's ground-state structure.     

Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes

The pineal gland has proven to be an excellent model for the study of adrenergic control systems. Noradrenaline, released from sympathetic nerve terminals in the pineal gland, regulates a large nocturnal increase in melatonin synthesis by stimulating the activity of arylalkylamine N-acetyltransferase (NAT, EC 2.3.1.87) 30-70-fold. An essential step in both the induction and maintenance of high NAT activity is an increase in intracellular cyclic AMP. Noradrenaline acts via beta-adrenoceptors to increase pineal cyclic AMP by activating adenylate cyclase, and the activation of pineal alpha 1-adrenoceptors potentiates beta-adrenergic stimulation not only of NAT but of both cyclic AMP and cyclic GMP. Here we describe investigations designed to test whether alpha 1-adrenergic potentiation of beta-adrenergic stimulation of pineal cyclic AMP involves protein kinase C. Our results suggest that kinase activation is involved and the data provide the first demonstration of a synergistic interaction between Ca2+-phospholipid-dependent protein kinase (protein kinase C) and neurotransmitter-dependent stimulation of cyclic AMP.     

Dependence of Z-DNA antibody binding to polytene chromosomes on acid fixation and DNA torsional strain

There is considerable interest in the existence and significance of alternative conformations of DNA to the right-handed B-form described originally by Watson and Crick. The indirect immunofluorescence observations of Nordheim et al., Arndt-Jovin et al. and Lemeunier et al. that antibodies against left-handed Z-DNA bind to polytene chromosomes have thus assumed considerable importance. However, there is a paradox: some workers observe Z-DNA in interbands and others in bands. We report here that binding of Z-DNA antibodies to Drosophila polytene chromosomes prepared without acid fixation is at background level, and that following acid fixation the same antibody treatment leads to intense fluorescence. Depending on the extent of exposure to 45% acetic acid, fluorescence can occur primarily in interbands or in bands. Furthermore, antibody binding is dependent on elastic torsional strain in the DNA molecules.     

核酸适配体氯化血红素比色法检测牛奶中的氯霉素

利用一种基于适配体氯化血红素(hemin)比色分析法快速检测牛奶中的氯霉素(chloram-phenicol,CAP)含量.设计两条DNA链,其中一条为CAP的核酸适配体,在其3′端修饰氯化血红素;另一条为CAP核酸适配体的互补链(cDNA),在其5′端修饰hemin.当体系中无CAP时,两条DNA单链杂交形成DNA双...     

Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster

Cyclic AMP is an intracellular mediator ('second messenger') in the nervous and endocrine control of cellular function, regulating different processes in different cell types. Although evidence is incomplete, it seems that cyclic AMP enhances the calcium-mediated release of neurotransmitter in some neurones. A simple form of memory in the mollusc Aplysia is probably encoded as a cyclic AMP-induced enhancement of neurotransmission at certain synapses of the central nervous system. The possibility that cyclic AMP participates in learning mechanisms may be explored using genetic mutants. For this purpose the fruitfly Drosophila is suitable as it is genetically well characterized and can learn through olfaction, vision or taste. We show here that independent searches for mutations of olfactory learning and of cyclic AMP metabolism, and for mutations causing female infertility have each led to the same gene--the dunce gene. Our evidence indicates that the normal dunce gene may specify a cyclic AMP phosphodiesterase.     

Crystal structure of the heterotrimer core of Saccharomyces cerevisiae AMPK homologue SNF1

AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals and is an attractive target for drug discovery against diabetes, obesity and other diseases. The AMPK homologue in Saccharomyces cerevisiae, known as SNF1, is essential for responses to glucose starvation as well as for other cellular processes, although SNF1 seems to be activated by a ligand other than AMP. Here we report the crystal structure at 2.6 A resolution of the heterotrimer core of SNF1. The ligand-binding site in the gamma-subunit (Snf4) has clear structural differences from that of the Schizosaccharomyces pombe enzyme, although our crystallographic data indicate that AMP can also bind to Snf4. The glycogen-binding domain in the beta-subunit (Sip2) interacts with Snf4 in the heterotrimer but should still be able to bind carbohydrates. Our structure is supported by a large body of biochemical and genetic data on this complex. Most significantly, the structure reveals that part of the regulatory sequence in the alpha-subunit (Snf1) is sequestered by Snf4, demonstrating a direct interaction between the alpha- and gamma-subunits and indicating that our structure may represent the heterotrimer core of SNF1 in its activated state.     

The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals

Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. Here we show that the SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signalling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibres. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signalling complexes.     

Role of poly(ADP-ribose) formation in DNA repair.

The abundant nuclear enzyme poly(ADP-ribose) polymerase catalyses the synthesis of poly(ADP-ribose) from nicotinamide adenine dinucleotide (NAD+). This protein has an N-terminal DNA-binding domain containing two zinc-fingers, which is linked to the C-terminal NAD(+)-binding domain by a short region containing several glutamic acid residues that are sites of auto-poly(ADP-ribosyl)ation. The intracellular production of poly(ADP-ribose) is induced by agents that generate strand interruptions in DNA. The branched homopolymer chains may attain a size of 200-300 residues but are rapidly degraded after synthesis. The function of poly(ADP-ribose) synthesis is not clear, although it seems to be required for DNA repair. Here we describe a human cell-free system that enables the role of poly(ADP-ribose) synthesis in DNA repair to be characterized. The results indicate that unmodified polymerase molecules bind tightly to DNA strand breaks; auto-poly(ADP-ribosyl)ation of the protein then effects its release and allows access to lesions for DNA repair enzymes.     

Role of cyclic AMP in a serotonin-evoked slow inward current in snail neurones

One model of synaptic transmission suggests that transmitters modify postsynaptic permeability through the intermediary of cyclic AMP. Thus, serotonin (5-hydroxytryptamine) evokes in molluscan neurones a decrease in a voltage-dependent K+ conductance which in turn generates a slow inward current when studied in steady voltage-clamp conditions. The serotonin-induced increase of the plateau phase of the spike of an Aplysia sensory neurone can be mimicked by both intracellularly injected cyclic AMP and extracellularly applied phosphodiesterase inhibitors, suggesting that cyclic AMP mediates the effect. We have tested whether a similar mechanism could account for the serotonin slow inward current in identified snail neurones and have found that the intracellular injection of cyclic AMP, but not of cyclic GMP or 5'-AMP, evokes a slow inward current showing similar voltage dependence, inversion potential and ionic properties to the serotonin slow inward current. Phosphodiesterase inhibitors at low concentrations (1-20 microM) potentiate the serotonin slow inward current and at higher concentrations evoke by themselves an inward current, partially or totally occluding the serotonin and cyclic AMP currents. Finally, we have found that in homogenates of pooled identified snail neurones serotonin stimulates the adenylate cyclase, increasing its activity by 50-100%.     

Left-handed helical conformation of poly[d(A-m5C).d(G-T)

Poly[d(G-C)] serves as the prototype for the right-to-left (B to Z) transition in he helical sense of DNA, both in solution and inthe crystal form. However, the question remains as to which other synthetic and natural DNAs have the potential to adopt left-handed conformations. One logical candidate is the canonical alternating purine-pyrimidine sequence d(A-C)n.d(G-T)n which seems to be widely disseminated in eukaryotic genomes. Our approach to this question is based on the enzymatic synthesis of poly[d(A-C).d(G-U)] derivatives with systematic methyl and halogen substitutions in the C-5 position of the pyrimidines C and U. Such modifications in poly[d(G-C)] have previously been shown to potentiate the B to Z transition. Here we report a highly cooperative, reversible, salt- and temperature-dependent transition for poly[d(A-m5C).d(G-T)], a repeat of the d(A-m5C) sequence which may occur in natural DNA. Spectroscopic studies and the demonstrated ability to bind anti-Z-DNA antibodies suggest that the new helical conformation is left-handed and shares structural features with known Z-DNA. However, a novel property, not exhibited by poly[d(G-C)], is the profound temperature dependence of the conformational equilibrium.     

Use of an 125I-labelled DNA ligand to probe DNA structure

It no longer seems likely that DNA molecules in situ have a uniform conformation, represented by the classical B-form helix. For example, recent structural studies have shown that in certain conditions DNA can have a left-handed (so-called Z-form) helix, and have revealed extensive sequence-dependent variations of B-DNA helical parameters. Such sequence-dependent variations in DNA structure can be investigated in solution with reagents that bind to DNA in a conformation-dependent manner, and cut one or both strands of the double-helix at the site of binding, as, for example, has been shown for the endonuclease DNase I3. We describe here a simple way to endow a DNA-binding ligand with the ability to cleave DNA--labelling with 125I. The radiochemical damage associated with 125I decay induces a double-stranded DNA break. Using this technique we have shown that a sequence of four consecutive A X T base pairs is a necessary, but not sufficient, condition for strong binding to DNA of the bis-benzamide Hoechst 33258--presumably the other important factor is the conformation of the double-helix at the site of the (A/T)4 sequence. We suggest 125I-Hoechst 33258 may be a useful new probe of DNA structure.