Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods.

In vertebrate photoreceptors, light reduces cyclic GMP concentration and closes cGMP-activated channels to induce a hyperpolarizing response. As Ca2+ can permeate the channels and the Na(+)-Ca2+ exchanger continuously extrudes Ca2+, closure of the channel results in a reduction of the inter-rod Ca2+ concentration. This is believed to be one of the mechanisms of light-adaptation produced by activation of guanylate cyclase. Effects of Ca2+ on the cGMP phosphodiesterase (PDE) have been reported, but their physiological significance has remained unclear. We have perfused the inside-out preparation of a frog rod outer segment (I/O ROS, originally termed truncated ROS, and find that Ca2+ in a physiological range regulates the light-activation of PDE. Therefore, PDE regulation by Ca2+ must be involved in light-adaptation in rods. The effect is mediated by a newly found protein which binds to disk membranes at high Ca2+ concentrations and prolongs PDE activation.     

Endothelium-dependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation

The action of some vascular smooth muscle relaxants depends on the presence of the endothelium. We have recently shown that relaxation may be mediated through the formation of cyclic GMP. The nitrovasodilators are another class of relaxants which exert their effects through the formation of cyclic GMP, although their relaxation is independent of the presence of the endothelium. Their relaxant properties seem to depend on free radical formation--specifically, the formation of nitric oxide. The NO-induced smooth muscle relaxation is proposed to occur through activation of guanylate cyclase and the formation of cyclic GMP. Protein phosphorylation is thought to be a common event in the pathway for many biological phenomena. Moreover, sodium nitroprusside and 8-bromo cyclic GMP induce similar patterns of protein phosphorylation in intact rat thoracic aorta. Here we report that the patterns of protein phosphorylation induced by the endothelium-dependent vasodilators and nitrovasodilators were identical. Incorporation of 32P into myosin light chain was decreased by both classes of agents. Removal of the endothelium abolished the changes in phosphorylation with the endothelium-dependent vasodilator (acetylcholine), but not those with the nitrovasodilator (sodium nitroprusside). These results suggest that endothelium-dependent vasodilators and nitrovasodilators induce relaxation through cyclic GMP-dependent protein phosphorylation and dephosphorylation of myosin light chain.     

Energy-dependent regulation of cell structure by AMP-activated protein kinase

AMP-activated protein kinase (AMPK, also known as SNF1A) has been primarily studied as a metabolic regulator that is activated in response to energy deprivation. Although there is relatively ample information on the biochemical characteristics of AMPK, not enough data exist on the in vivo function of the kinase. Here, using the Drosophila model system, we generated the first animal model with no AMPK activity and discovered physiological functions of the kinase. Surprisingly, AMPK-null mutants were lethal with severe abnormalities in cell polarity and mitosis, similar to those of lkb1-null mutants. Constitutive activation of AMPK restored many of the phenotypes of lkb1-null mutants, suggesting that AMPK mediates the polarity- and mitosis-controlling functions of the LKB1 serine/threonine kinase. Interestingly, the regulatory site of non-muscle myosin regulatory light chain (MRLC; also known as MLC2) was directly phosphorylated by AMPK. Moreover, the phosphomimetic mutant of MRLC rescued the AMPK-null defects in cell polarity and mitosis, suggesting MRLC is a critical downstream target of AMPK. Furthermore, the activation of AMPK by energy deprivation was sufficient to cause dramatic changes in cell shape, inducing complete polarization and brush border formation in the human LS174T cell line, through the phosphorylation of MRLC. Taken together, our results demonstrate that AMPK has highly conserved roles across metazoan species not only in the control of metabolism, but also in the regulation of cellular structures.     

Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes

In heart cells, cyclic AMP-dependent protein kinase (PKA) regulates calcium- and potassium-ion current by phosphorylating the ion channels or closely associated regulatory proteins. We report here that isoprenaline induced large chloride-ion currents in voltage-clamped, internally-dialysed myocytes from guinea-pig ventricles. The Cl- current could be activated by intracellular dialysis with cAMP or the catalytic subunit of PKA, indicating regulation by phosphorylation. In approximately symmetrical solutions of high Cl- concentration, the macroscopic cardiac Cl- current showed little rectification, unlike the single-channel current in PKA-regulated Cl- channels of airway epithelial cells. But, like epithelial Cl- -channel currents, the cardiac Cl- current was sensitive to the distilbene,4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS). In the absence of kinase activation, cardiac sarcolemmal Cl- conductance was negligible. During beta-adrenergic stimulation of the heart, this novel Cl- conductance should accelerate action-potential repolarization and so protect impulse propagation in the face of the possibly arrhythmogenic increases in heart rate and in calcium entry into the cells.     

Prevention of natural motoneurone cell death by dibutyryl cyclic GMP

Natural neuronal cell death is a well-described developmental phenomenon common to many nerve centres in a variety of animal species. Neuronal survival has been shown to depend on the presence and size of the available target tissue and it has been suggested that neuronal survival is dependent on successful competition for either a limited number of synaptic sites or a limited amount of trophic factor(s). In the lateral motor column of the lumbar spinal cord in the chick embryo, the period of axon elongation and innervation of the periphery has been shown to precede that of natural motoneurone cell death. While muscle contractile activity appears to regulate the extent of motoneurone death, to date the intracellular molecular events that initiate and regulate the developmental process of natural neuronal cell death or, more importantly, neuronal survival are unknown. Our earlier studies suggested that either contact or association between spinal cord processes and muscle cells during neuromuscular junction formation in vivo leads to an increase in cyclic GMP in whole spinal cord. We now show that treatment of chick embryos with the membrane-permeable cyclic GMP analogue, dibutyryl cyclic GMP during the period of natural motoneurone cell death prevents greater than 58% of natural motoneurone cell death in the lumbar lateral motor column.     

Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR.

Cystic fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability, increased mucous sulphation, increased Na+ absorption and defective acidification of lysosomal vesicles. The CF gene encodes a protein (the cystic fibrosis transmembrane conductance regulator, CFTR) that can function as a low-conductance Cl- channel with a linear current-voltage relationship whose regulation is defective in CF patients. Larger conductance, outwardly rectifying Cl- channels are also defective in CF and fail to activate when exposed either to cyclic AMP-dependent protein kinase A or to protein kinase C. The role of the outwardly rectifying Cl- channel in CF has been questioned. We report here that expression of recombinant CF genes using adeno-associated virus vectors in CF bronchial epithelial cells corrects defective Cl- secretion, that it induces the appearance of small, linear conductance Cl- channels, and restores protein kinase A activation of outwardly rectifying Cl- channels. These results re-establish an involvement of outwardly rectifying Cl- channels in CF and suggest that CFTR regulates more than one conductance pathway in airway tissues.     

Control of Ca2+ in rod outer segment disks by light and cyclic GMP

Photons absorbed in vertebrate rods and cones probably cause electrochemical changes at the photoreceptor plasma membrane by changing the cytoplasmic concentration of a diffusible transmitter substance, reducing the Na+ current flowing into the outer segment of the cell in the dark, to produce the observed membrane hyperpolarization that is the initial excitatory response. Cyclic GMP has been proposed as the transmitter because a light-activated cyclic GMP phosphodiesterase (PDE) has been found in rod disk membranes and because intracellularly injected cyclic GMP reduces rod membrane potentials. Free Ca2+ has also been proposed because increasing external [Ca2+] quickly and reversibly reduces the dark current and divalent cationophores increase the Ca2+ sensitivity. Ca2+ efflux from rod outer segments (ROS) of intact retinas occurs simultaneously with light responses. Vesicles prepared from ROS disk membranes become more permeable on illumination, releasing trapped ions or molecules, but intact outer segment disks have not previously been found to store sufficient Ca2+ in darkness and to release enough in light to meet the theoretical requirements for control of the dark current by varying cytoplasmic Ca2+ (refs 14-18). We now report experiments that show the required Ca2+ storage and release from rod disk membranes suspended in media containing high-energy phosphate esters and electrolytes approximating the cytoplasmic composition of live rod cells. Cyclic GMP stimulates Ca2+ uptake by ROS disks in such media.     

一个水稻蛋白激酶的过表达分析及表达调控

对一个预测的具有光反应活性的丝氨酸/苏氨酸蛋白激酶STK进行过表达研究,构建了STK过表达载体,并用农杆菌介导的方法进行遗传转化,获得T0代转基因植株.对转基因阳性植株进行表达分析的结果表明,转基因植株STK基因的表达量显著高于对照,说明目的基因得到了过表达.另外,利用获得的过表达转基因植株对STK调控水稻中光周期相关的开花基因Hd1和Hd3a的表达进行分析,结果表明,Hd1及Hd3a的表达在转基因植株中明显上调,表明该蛋白激酶的基因可能位于Hd1和Hd3a上游,对于Hd1和Hd3a的表达具有重要的调控作用.     

Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.

Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrP(Sc), which is a conformationally modified isoform of a normal cell-surface protein called PrP(C) (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrP(Sc) replicate by conversion of host PrP(C), generating large amounts of PrP(Sc) aggregates in the brains of diseased individuals. We aimed to reproduce this event in vitro. Here we report a procedure involving cyclic amplification of protein misfolding that allows a rapid conversion of large excess PrP(C) into a protease-resistant, PrP(Sc)-like form in the presence of minute quantities of PrP(Sc) template. In this procedure, conceptually analogous to polymerase chain reaction cycling, aggregates formed when PrP(Sc) is incubated with PrP(C) are disrupted by sonication to generate multiple smaller units for the continued formation of new PrP(Sc). After cyclic amplification more than 97% of the protease-resistant PrP present in the sample corresponds to newly converted protein. The method could be applied to diagnose the presence of currently undetectable prion infectious agent in tissues and biological fluids, and may provide a unique opportunity to determine whether PrP(Sc) replication results in the generation of infectivity in vitro.     

Opposite effects of cyclic GMP and cyclic AMP on Ca2+ current in single heart cells

The slow inward Ca2+ current, ICa, is fundamental in the initiation of cardiac contraction and neurohormonal regulation of cardiac function. It is increased by beta-adrenergic agonists, which stimulate synthesis of cyclic AMP (cAMP) and cAMP-dependent phosphorylation. The neurotransmitter acetylcholine reduces ICa by an unknown mechanism. There is strong evidence that acetylcholine reduces ICa by decreasing adenylate cyclase activity, but cGMP has also been implicated as ACh stimulates cGMP accumulation and activates cGMP-dependent protein kinase. Application of cGMP decreases contractile force, decreases Ca flux, shortens the duration of action potentials and inhibits Ca-dependent action potentials. Other studies, however, have concluded that cGMP levels do not correlate with contractile force and that cGMP has no effect on ICa. We have therefore examined the effects of intracellular perfusion of cGMP on ICa using isolated, voltage-clamped cells from frog ventricle. We find that cGMP has negligible effects on basal ICa, but greatly decreases the ICa that had been elevated by beta-adrenergic agonists or by intracellular perfusion with cAMP. The decrease of ICa is mediated by cAMP hydrolysis via a cGMP-stimulated cyclic nucleotide phosphodiesterase.     

Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment

Vertebrate rod photoreceptors hyperpolarize when illuminated, due to the closing of cation-selective channels in the plasma membrane. The mechanism controlling the opening and closing of these channels is still unclear, however. Both 3',5'-cyclic GMP and Ca2+ ions have been proposed as intracellular messengers for coupling the light activation of the photopigment rhodopsin to channel activity and thus modulating light-sensitive conductance. We have now studied the effects of possible conductance modulators on excised 'inside-out' patches from the plasma membrane of the rod outer segment (ROS), and have found that cyclic GMP acting from the inner side of the membrane markedly increases the cationic conductance of such patches (EC50 30 microM cyclic GMP) in a reversible manner, while Ca2+ is ineffective. The cyclic GMP-induced conductance increase occurs in the absence of nucleoside triphosphates and, hence, is not mediated by protein phosphorylation, but seems rather to result from a direct action of cyclic GMP on the membrane. The effect of cyclic GMP is highly specific; cyclic AMP and 2',3'-cyclic GMP are completely ineffective when applied in millimolar concentrations. We were unable to recognize discrete current steps that might represent single-channel openings and closings modulated by cyclic GMP. Analysis of membrane current noise shows the elementary event to be 3 fA with 110 mM Na+ on both sides of the membrane at a membrane potential of -30 mV. If the initial event is assumed to be the closure of a single cyclic GMP-sensitive channel, this value corresponds to a single-channel conductance of 100 fS. It seems probable that the cyclic GMP-sensitive conductance is responsible for the generation of the rod photoresponse in vivo.     

MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin.

AGONISTS that stimulate protein kinase C (PKC) induce profound changes in cell morphology correlating with the reorganization of submembranous actin, but no direct connection between PKC and actin assembly has been identified. The myristoylated, alanine-rich C kinase substrate (MARCKS) binds calmodulin and is a predominant, specific substrate of PKC which is phosphorylated during macrophage and neutrophil activation , growth factor-dependent mitogenesis and neurosecretion; it is redistributed from plasma membrane to cytoplasm when phosphorylated and is involved in leukocyte motility. Here we report that MARCKS is a filamentous (F) actin crosslinking protein, with activity that is inhibited by PKC-mediated phosphorylation and by binding to calcium-calmodulin. MARCKS may be a regulated crossbridge between actin and the plasma membrane, and modulation of the actin crosslinking activity of the MARCKS protein by calmodulin and phosphorylation represents a potential convergence of the calcium-calmodulin and PKC signal transduction pathways in the regulation of the actin cytoskeleton.     

The molecular heterogeneity of protein kinase C and its implications for cellular regulation

Protein kinase C is now known to be a large family of proteins, with multiple subspecies that have subtle individual enzymological characteristics. Some members of the family exhibit distinct patterns of tissue expression and intracellular localization; different kinases probably have distinct functions in the processing and modulation of a variety of physiological and pathological responses to external signals.     

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.     

Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase.

Leptin is a hormone secreted by adipocytes that plays a pivotal role in regulating food intake, energy expenditure and neuroendocrine function. Leptin stimulates the oxidation of fatty acids and the uptake of glucose, and prevents the accumulation of lipids in nonadipose tissues, which can lead to functional impairments known as "lipotoxicity". The signalling pathways that mediate the metabolic effects of leptin remain undefined. The 5'-AMP-activated protein kinase (AMPK) potently stimulates fatty-acid oxidation in muscle by inhibiting the activity of acetyl coenzyme A carboxylase (ACC). AMPK is a heterotrimeric enzyme that is conserved from yeast to humans and functions as a 'fuel gauge' to monitor the status of cellular energy. Here we show that leptin selectively stimulates phosphorylation and activation of the alpha2 catalytic subunit of AMPK (alpha2 AMPK) in skeletal muscle, thus establishing a previously unknown signalling pathway for leptin. Early activation of AMPK occurs by leptin acting directly on muscle, whereas later activation depends on leptin functioning through the hypothalamic-sympathetic nervous system axis. In parallel with its activation of AMPK, leptin suppresses the activity of ACC, thereby stimulating the oxidation of fatty acids in muscle. Blocking AMPK activation inhibits the phosphorylation of ACC stimulated by leptin. Our data identify AMPK as a principal mediator of the effects of leptin on fatty-acid metabolism in muscle.     

Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites

p60src, the transforming protein of Rous sarcoma virus (RSV), is a protein kinase that has a strict specificity for tyrosine. The phosphorylation of cellular proteins by p60src (ref. 4) results in transformation. Recently, Barker and Dayhoff discovered that residues 259-485 of p60src have 22% sequence identity with residues 33-258 of the catalytic subunit of cyclic AMP-dependent protein kinase, an enzyme that has a specificity for serine. Because it was necessary to introduce eight gaps to align the two proteins, the question remained as to whether this apparent homology reflected a common evolutionary origin. We demonstrate here that the ATP analogue p-fluorosulphonylbenzoyl 5'-adenosine (FSBA) inactivates the tyrosine protein kinase activity of p60src by reacting with lysine 295. When aligned for maximum sequence identity, lysine 295 of p60src and the lysine in the catalytic subunit which also reacts specifically with FSBA are superimposed precisely. This functional homology is strong evidence that the protein kinases, irrespective of amino acid substrate specificity, comprise a single divergent gene family.