Cyclic AMP-dependent Protein Phosphorylation Research Articles

Iron Downregulates Leptin by Decreasing CREB O-GlcNAc Modification and CREB Occupancy of the Leptin Promoter

Modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) serves as a nutrient-dependent signal to regulate transcription of the leptin gene. Recently we reported that increased levels of iron, which are also a risk factor for type 2 diabetes, down regulate leptin in mice fed high iron diets (2000 mg/kg) or in 3T3L1 cells treated with iron. Iron increased cyclic AMP-dependent response element binding protein (CREB) phosphorylation and its occupancy at inhibitory sites in the leptin promoter, but without demonstrable increases in CREB kinase activities. We now demonstrate that nutrient- and iron-dependent regulation of leptin are linked through O-GlcNAc modification of CREB. CREB O-GlcNAc modification was decreased in mice fed high iron, and this was associated with 54±7% and 44±9% reductions in recruitment to its two sites in the leptin promoter as analyzed by ChIP (p<0.05). Increasing levels of O-GlcNAc through targeted deletion of the O-GlcNAcase (OGA) gene in adipocytes increased adipose leptin mRNA expression by 86±12% (p<0.05) in mice fed a low-normal iron diet (35 mg/kg) and partially rescued the effect of high iron (35±3%, p<0.05). Similar effects were seen in serum leptin levels: High iron caused a 72% decrease and OGA deletion resulted in a rescue of 34%. Treatment of 3T3L1 cells with iron decreased expression of a leptin reporter plasmid by 45±4% and glucosamine, which bypasses the rate-limiting step in synthesis of UDP-GlcNAc, the substrate for O-GlcNAcylation, increased protein O-GlcNAc and fully rescued (106±5%) the effect of high iron (p<0.001 for both). Although iron also decreased global levels of protein O-GlcNAc modification, mRNA levels of the enzymes that control the modification, namely O-GlcNAc transferase and OGA, were unchanged. We conclude that adipose tissue integrates cellular nutrient status and tissue iron levels to control the regulation of leptin, through regulation of O-GlcNAc modification of the transcription factor CREB. Disclosure J. Liu: None. Y. Gao: None. F. Lorenzo: None. S.T. Sink: None. D. McClain: None.

Fractalkine/CX3CL1 engages different neuroprotective responses upon selective glutamate receptor overactivation.

Neuronal death induced by overactivation of N-methyl-d-aspartate receptors (NMDARs) is implicated in the pathophysiology of many neurodegenerative diseases such as stroke, epilepsy and traumatic brain injury. This toxic effect is mainly mediated by NR2B-containing extrasynaptic NMDARs, while NR2A-containing synaptic NMDARs contribute to cell survival, suggesting the possibility of therapeutic approaches targeting specific receptor subunits. We report that fractalkine/CX3CL1 protects hippocampal neurons from NMDA-induced cell death with a mechanism requiring the adenosine receptors type 2A (A2AR). This is different from CX3CL1-induced protection from glutamate (Glu)-induced cell death, that fully depends on A1R and requires in part A3R. We show that CX3CL1 neuroprotection against NMDA excitotoxicity involves D-serine, a co-agonist of NR2A/NMDAR, resulting in cyclic AMP-dependent transcription factor cyclic-AMP response element-binding protein (CREB) phosphorylation.

Cyclic AMP-elevating agents prevent oligodendroglial excitotoxicity.

Previously, we have demonstrated that cells of the oligodendroglial lineage express non-NMDA glutamate receptor genes and are damaged by kainate-induced Ca2+ influx via non-NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte-like cells, differentiated from the CG-4 cell line established from rat oligodendrocyte type-2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate-induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8-bromo-cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate-induced cell death in an additive manner. A remarkable increase in Ca2+ influx (approximately 5.5-fold) also was induced by kainate. The cyclic AMP-elevating agents caused a partial suppression of the kainate-induced increase in Ca2+ influx, leading to a less prominent response of intracellular Ca2+ concentration to kainate. The suppressing effect of forskolin on the kainate-induced Ca2+ influx was partially reversed by H-89, an inhibitor of cyclic AMP-dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate-induced Ca2+ influx. We therefore concluded that cyclic AMP-elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP-dependent protein kinase-dependent protein phosphorylation, resulting in decreased kainate-induced Ca2+ influx.

Involvement of altered cytoskeletal protein phosphorylation in aluminum-induced CNS dysfunction.

In the present investigation, we have studied the effects of aluminum (10 mg/kg of body weight per day, i.p.) and desferrioxamine (6 mg/kg of body weight per day, i.p.) alone and in combination for 4 weeks on the regulation of phosphorylation of neuronal proteins. A marked decrease in the biological activity of calmodulin was observed after aluminum treatment; however, in combination with desferrioxamine, a reversal in the levels of calmodulin, in terms of nmol cAMP hydrolyzed/min/mg protein, was observed. Exogenous addition of calmodulin had an inhibitory effect on calmodulin-mediated synaptosomal protein phosphorylation in aluminum-exposed animals. An almost complete reversal of this inhibition was observed following coexposure to aluminum and desferrioxamine. Cyclic AMP-dependent synaptosomal protein phosphorylation was, however, stimulated following aluminum exposure. Coadministration of desferrioxamine along with aluminum was found to mitigate the neurotoxic effect of aluminum.

Stimulation of Calcium Influx and Calcium Cascade by Cyclic AMP in Cultured Carrot Cells

Treatment of cultured carrot (Daucus carota L.) cells with activators of adenylate cyclase, forskolin, and cholera toxin induced the biosynthesis of an antifungal isocoumarin, 6-methoxymellein, in the cells. Addition of dihutyryl cyclic AMP to carrot cell culture also stimulated the accumulation of the compound. The cyclic AMP-evoked 6-methoxymellein production was significantly depressed in the presence of certain inhibitors of calcium cascade such as Ca2+ channel blockers and inhibitors of calmodulin-dependent processes. In dibutyryl cyclic AMP- and forskolin-treated carrot cells, increase in cytosolic Ca2+ concentration was observed as monitored by the fluorescent calcium indicator fluo-3. Cyclic AMP-dependent Ca2+ influx into carrot cells was also confirmed with Ca2+-loaded vesicles prepared from the plasma membrane-rich fraction of the cells. Transient increase in Ca2+- and Ca2+/calmodulin-dependent protein kinase activity but not cyclic AMP-dependent protein phosphorylation was detected in the cells of high cyclic AMP concentration. Results obtained in the present work suggest that the increase in cyclic AMP content in carrot cells induces Ca2+ influx across plasma membrane without activating cyclic AMP-dependent protein kinase which, then, stimulates calcium cascade in the cells.

Chronic administration of lithium or other antidepressants increases levels of DARPP-32 in rat frontal cortex.

We studied the chronic actions of lithium on rat brain by investigating its effects on cyclic AMP-dependent protein phosphorylation by use of a back-phosphorylation procedure. We identified one heavily regulated phosphoprotein in frontal cortex as the 32-kDa dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32). Immunoblot experiments demonstrated that chronic lithium regulation of DARPP-32 back-phosphorylation is associated with equivalent increases in levels of DARPP-32 immunoreactivity. Lithium regulation of DARPP-32 immunoreactivity required chronic drug administration and was not observed in several other brain regions examined. Moreover, chronic administration of the antidepressant imipramine or tranylcypromine produced a similar increase in levels of DARPP-32 in frontal cortex, whereas other types of psychotropic drugs, including haloperidol, morphine, and cocaine, did not influence DARPP-32 levels. Increased levels of DARPP-32 could reflect a common functional effect on frontal cortex of long-term exposure to lithium and some other antidepressant medications, an effect possibly related to the clinical actions of these drugs.

Ca2(+)-induced insulin secretion from electrically permeabilized islets. Loss of the Ca2(+)-induced secretory response is accompanied by loss of Ca2(+)-induced protein phosphorylation.

Increasing the cytosolic Ca2+ concentration of electrically permeabilized rat islets of Langerhans caused rapid increases in insulin secretion and in 32P incorporation into islet proteins. However, the secretory responsiveness of permeabilized islets was relatively transient, with insulin secretion approaching basal levels within 20-30 min despite the continued presence of stimulatory concentrations of Ca2+. The loss of Ca2(+)-induced insulin secretion was accompanied by a marked reduction in Ca2(+)-dependent protein phosphorylation, but not in cyclic AMP-dependent protein phosphorylation. Similarly, permeabilized islets which were no longer responsive to Ca2+ were able to mount appropriate secretory responses to cyclic AMP and to a protein kinase C-activating phorbol ester. These results suggest that prolonged exposure to elevated cytosolic Ca2+ concentrations results in a specific desensitization of the secretory mechanism to Ca2+, perhaps as a result of a decrease in Ca2(+)-dependent kinase activity. Furthermore, these studies suggest that secretory responses of B-cells to cyclic AMP and activators of protein kinase C are not dependent upon the responsiveness of the cells to changes in cytosolic Ca2+.

The plasma membrane ferrireductase activity of Saccharomyces cerevisiae is partially controlled by cyclic AMP.

The plasma-membrane-bound ferrireductase activity of ras1 and ras2 mutants of Saccharomyces cerevisiae is not induced in response to iron limitation. This phenotype was suppressed by the bcy1 mutation in ras2 but not in ras1 mutants. The cellular haem content of ras-1-bearing strains decreased dramatically when cells were grown in semi-synthetic medium (low yeast extract content), which could account for their very low ferrireductase activity. The ferrireductase activity of cdc25 and cdc35 mutants dropped when the cells were shifted to a non-permissive temperature. This drop was prevented in the double mutant cdc35 sra5 by adding cyclic AMP to the growth medium. We propose that ferrireductase activity is under the control of a cyclic AMP-dependent protein phosphorylation.

Identification and localization of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator.

Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland.

Dopaminergic brain reward regions of Lewis and Fischer rats display different levels of tyrosine hydroxylase and other morphine- and cocaine-regulated phosphoproteins

We studied cyclic AMP-dependent protein phosphorylation in the mesolimbic and nigrostriatal dopamine systems of two genetically inbred rat strains, Lewis (LEW) and Fischer (F344) rats. These strains represent genetically divergent populations of rats that have been used to study possible genetic factors involved in a variety of biological processes. We found striking differences in levels of tyrosine hydroxylase, and several other phosphoproteins, in the mesolimbic, but not the nigrostriatal, dopamine system between the two rat strains. Interestingly, in Sprague-Dawley rats, these same phosphoproteins are altered by chronic morphine and chronic cocaine specifically in the mesolimbic dopamine system, generally thought to be a brain reward pathway that mediates some of the reingorcing actions of many drugs of abuse. As LEW and F344 rats have been reported to show different levels of preference for several types of drugs of abuse, the results are consistent with the possibility that these phosphoproteins may mediate aspects of drug reinforcement and contribute to individual differences in vulnerability to drug addiction.

Catabolite inactivation, cyclic AMP and protein phosphorylation in the methylotrophic yeastHansenula polymorpha

The inactivation of the peroxisomal enzyme alcohol oxidase and the cytoplasmic enzymes fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase was found to occur after addition of glucose to methanol-grown cells of the yeast Hansenula polymorpha. The concentration of cyclic AMP increased nearly twofold within 3 min under the same conditions. In crude extracts of H. polymorpha about 20 proteins are phosphorylated by cyclic AMP dependent protein kinases, among them also fructose-1,6-bisphosphatase. No phosphorylation of the alcohol oxidase protein could be detected. From this fact, it was concluded that the inactivation of the peroxisomal alcohol oxidase is independent of cyclic AMP-dependent protein phosphorylation.

Effects of chronic lithium treatment on protein kinase c and cyclic AMP-dependent protein phosphorylation

Lithium inhibits the agonist-induced hydrolysis of phosphoinositides and the synthesis of cyclic AMP (cAMP) in rat brain preparations, each of which is linked to activation of specific protein kinases. Therefore, we examined the effects of chronic lithium treatment on protein kinase activities in rat hippocampus. Chronic lithium treatment did not alter the distribution or activity of protein kinase C in hippocampal soluble or particulate fractions. However, chronic lithium treatment increased protein kinase C-mediated phosphorylation of four endogenous proteins in the soluble fraction (16,17,20,22 kD) and reduced the phosphorylation of three proteins (18,19,87 kD) in the particulate fraction. Chronic lithium treatment did not alter cAMP-dependent phosphorylation of endogenous proteins in the soluble fraction but reduced phosphorylation of two proteins (54 and 71 kD) in the particulate fractions. These results demonstrate that besides inhibiting second messenger production in brain, chronic lithium treatment also causes specific alterations in the phosphorylation of endogenous proteins.

Cyclic AMP-dependent protein phosphorylation is involved in the development of negative slope resistance and a reduction of the potassium current induced by pentylenetetrazole in identified snail neurons

The role of cAMP-dependent protein phosphorylation in the pentylenetetrazole (PTZ)-induced bursting activity was examined in snail neurons, using the voltage clamp method in combination with the pressure injection technique. The cAMP-dependent protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and isoquinolinesulfonamide, inhibited the PTZ-induced negative slope resistance (NSR) in the steady state I– V curve. These inhibitors also suppressed the action of PTZ on the delayed outward potassium current ( I KD). This suppression was transiently abolished by intracellular injection of the catalytic subunit of cAMP-dependent protein kinase. These findings suggest that cAMP-dependent protein phosphorylation may be involved in both the development of the NSR and a reduction of the I KD by PTZ, leading to depolarizing phase of a bursting cycle.

Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition

Exposure of human platelets to prostacyclin (PGI2), iloprost or prostaglandin E1 (PGE1) elicits the cyclic AMP-dependent phosphorylation of proteins of 22, 24, 30, 39, 50, 60 and 250 kDa (P22, P24 etc.). P22 was recently identified as rap 1B, a ras-like protein, and P24 was shown to be the beta-chain of glycoprotein Ib. We found that cyclic AMP-dependent phosphorylation of all proteins except P22 was maximal 1 min after exposure of platelets to PGI2, iloprost or PGE1; maximal phosphorylation of P22 occurred after 45 min of incubation. Inhibition of thrombin-induced platelet activation required only a 30 s incubation with PGI2 or iloprost; at this time phosphorylation of P22 was only slightly increased. Although at maximal concentrations PGI2 was more potent than PGE1 in inhibiting thrombin-induced platelet activation, no difference in the degree and the kinetics of cyclic AMP-dependent protein phosphorylation was found. Platelets that had been preincubated and washed in the presence of PGE1 and later resuspended in the absence of PGE1 responded fully to activation by thrombin despite maximal phosphorylation of P22 and P24. Furthermore, addition of PGI2 to PGE1-washed platelets prevented thrombin-induced platelet activation, but did not evoke further phosphorylation of P22 or P24. Phosphorylation of P39 and P50 correlated better with PGI2-induced inhibition of platelet activation. In experiments in which PGE1-induced inhibition of platelet activation was overcome by the addition of thrombin, no dephosphorylation of proteins phosphorylated by cyclic AMP-dependent kinases was observed. These experiments indicate that: (a) phosphorylation of rap 1B and glycoprotein Ib is not related to platelet inhibition by cyclic AMP; (b) phosphorylation of other proteins such as P39 and P50 probably plays a role in mediating cyclic AMP-dependent platelet inhibition; (c) reactions other than cyclic AMP-dependent protein phosphorylation may participate in platelet inhibition by cyclic AMP.

Cyclic AMP-dependent membrane protein phosphorylation and calmodulin binding are involved in the rapid stimulation of muscle calcium uptake by 1,25-dihydroxyvitamin D3

Rapid in vivo effects of 1,25-dihydroxyvitamin D3 on muscle calcium metabolism have been reported. In vitro studies have shown that exposure of vitamin D-deficient chick soleus muscles to the sterol for 1-10 minutes causes a significant stimulation of tissue 45Ca uptake which can be suppressed by Ca channel blockers. A parallel increase in muscle membrane calmodulin content that could be mimicked by forskolin was observed. Experiments were carried out to obtain information about the mechanism underlying the fast action of 1,25-dihydroxyvitamin D3. Like the sterol, forskolin (10 microns) rapidly increased (+48% at 5 min) soleus muscle 45Ca uptake and its effect could be reversed by nifedipine (50 microns). In agreement with these observations, 1,25-dihydroxyvitamin D3 markedly elevated tissue cAMP levels within 45 seconds to 5 minutes of treatment in a dose-dependent manner (10(-11)-10(-7) M). Moreover, incubation of isolated muscle microsomes with 1,25-dihydroxyvitamin D3 increased adenylate cyclase activity and caused a similar profile of stimulation of protein phosphorylation with [gamma-32P]-ATP as forskolin. Major changes were detected in proteins whose calmodulin binding ability has been previously shown to be increased by 1,25-dihydroxyvitamin D3. In addition, the calmodulin antagonists fluphenazine and compound 48/80 abolished the increase in muscle Ca uptake and membrane calmodulin content produced by the sterol. The results suggest that 1,25-dihydroxyvitamin D3 activates muscle Ca channels through a direct membrane action which involves cAMP-dependent protein phosphorylation and calmodulin binding.

The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings

1. The importance of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP-dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated. 2. Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.p.f) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine. 3. The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8-(4-Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable. 4. All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release. 5. The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (30-100 microM) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel. 6. H7 prevented the increases in spontaneous ACh release produced by CPT cyclic AMP (250 microM). Thus H7 is likely to inhibit PK A in frog motor nerve endings. 7. H7 did not alter the inhibitory effect of adenosine on evoked and spontaneous ACh release. 8. The results suggest: (i) that the adenylyl cyclase-cyclic AMP-PK A system is compartmentalized within the motor nerve terminal, (ii) that phosphorylation does not play a major role in ACh release and (iii) the cyclic AMP-PK A system modulates rather than mediates the inhibitory effects of adenosine.

Cyclic AMP-dependent protein phosphorylation in primate kidney

T h e functions o f cell types within different tissues are coordinated by a variety of hormones. These increase the concentration o f second messengers, such as cyclic nucleotidcs, leading t o activation of protein kinases and phosphorylation o f specific proteins 11 I. T h e kidney is the target organ for a number of hormones which activate adenylate cyclase and hence cyclic AMP-dependent kinases [2 I. T h e purpose o f this study was to gain further understanding of the regulation o f kidney metabolism by measuring protein phosphorylation resulting from the action o f cyclic AMP-dependent protein kinases. Specific proteins phosphorylated were identified using electrophoresis and autoradiography. Studies were performed on cytosol and membrane preparations from primate kidney. Kidneys were obtained from recipient baboons undergoing transplant; normal human kidneys were donor kidneys which had not been used for transplant. All procedures were carried out at 4°C. Kidneys were decapsulated and outer cortex homogenized in sucrose medium. Membrane vesicles, including basolatcral and brush-border membranes, wcrc prepared using the method of Fitzpatrick et ul. [3] . Cytosol was obtained by ultracentrifugation ( 100 000 g, 1 h) o f the initial low-spin supernatant from the membrane preparation. Purity o f fractions was assessed using the marker enzymes glutamate dchydrogcnase, lactate dehydrogenase, Na +, K + adenosine triphosphatase and y-glutamyltransferase. After incubating tissue fractions with 0.1%) (v/v) 'Triton X-100 at 4°C for 1 0 min, phosphorylation was measured using a modification o f the assay of Szoka & Ettinger 14). Fractions (100-200 p g o f protein) were incubated with 40 mM-Tris/HCl, pH 7.2, 1 0 mM-MgC12 and 0.3 mM-EGTA in the presence or absence of 25 ,lAM-CyCliC A M P for 30 min at 4°C. After 1 min at 30°C the reaction was initiated by the addition o f 1 0 , ~ A M [ ~ ~ ~ P ) A T P (0.16 x 1 OJ d.p.m./pmol). T h e reaction was terminated after 5 min by the addition of icecold 25% (w/v) trichloroacetic acid. Protein was precipitated by centrifuging for 15 min at 800 g. T h e resulting pellet was washed three times in 25% (w/v) trichloroacetic acid, resuspended in I M-NaOH overnight and then counted in scintillant. For the identification o f phosphorylated proteins by means of electrophoresis and autoradiography, tissue fractions were treated as abovc except that the reaction was initiated by addition of 10 , ~ A M [ ~ ~ ~ P ] A T P (0.5 x 1 0 J d.p.m./ pmol) and incubation continued for 1 min at 30°C. T h e reaction was terminated by addition of 0.125 M-Tris/HCI, pH 6.8, 4%) (w/v) SDS, 20% (v/v) glycerol and 1 % (v/v) mercaptoethanol followed by boiling for 1 min. SDS/polyacrylamide-gel electrophoresis [ 5 ) employing a 4% stacking gel (2 em) and a 7.5% resolving gel (6 cm) was carried out at 15 mA/gel for 1.5 h. T h e gels were then dried and autoradiographed. Rainbow markers (Amcrsham, Bucks, U.K.) were used to estimated relative molecular mass. Phosphorylation occurred very rapidly ( < 1 min) and then remained constant for at least 20 min. Incorporation increased with increasing ATP concentrations from 1 0 to 250 ,LLM and then remained constant at 5 0 0 ~ M A T P . Phosphate incorporation after a 5 min incubation period was determined and expressed as pmol/mg of protein. In the absence of cyclic AMP, phosphate incorporation in baboon membrane was 30.7f 13.1 ( t i = 5 ) and in cytosol was 18.5 k 6 . 1 ( t i = 6 ) pmol/mg o f protein; in the presence of cyclic AMP, the respective values were 34.2+ 18.7 and 18.8 k 4.2 pmol/mg of protein. Fig. 1 shows an autoradiograph for baboon cytosol demonstrating that eight major proteins, of molecular mass in the range 31-135 kDa, were phosphorylated in the absence of cyclic AMP; three bands of mass 45, 55 and 66 kDa were intensified in the presence of cyclic AMP. Similar results were found for normal human kidney cytosol as

Inhibitory effect of a toxin okadaic acid, isolated from the black sponge on smooth muscle and platelets

1. Effects of okadaic acid, a toxin isolated from marine sponges, on smooth muscle contraction and platelet activation were examined. 2. Contractions in rabbit aorta induced by high concentrations of K+ and noradrenaline were inhibited by 0.1-1 microM okadaic acid in a concentration-dependent manner. Spontaneous rhythmic contractions as well as high K+-induced contraction in guinea-pig taenia caeci were also inhibited by 1 microM okadaic acid. 3. High K+-induced contraction in rabbit aorta was accompanied by increased Ca2+ influx measured with 45Ca2+ and increased cytosolic Ca2+ [( Ca2+]cyt) measured with fura-2-Ca2+ fluorescence. Okadaic acid inhibited the contraction without inhibiting Ca2+ influx and produced only a small decrease in [Ca2+]cyt. 4. In a saponin-skinned taenia, Ca2+-induced contraction was not inhibited but rather potentiated by okadaic acid. 5. Okadaic acid, 1 microM, inhibited aggregation, ATP release and increased in [Ca2+]cyt induced by thrombin in washed rabbit platelets. Okadaic acid itself did not change the platelet activities. 6. Okadaic acid did not change the cyclic AMP content of rabbit aorta although the inhibitory effects of okadaic acid were similar to those of cyclic AMP. 7. Although the mechanism of the inhibitory effect of okadaic acid was not clarified in the present experiments, it is suggested that okadaic acid acts by inhibiting protein phosphatases resulting in an indirect activation of cyclic AMP-dependent protein phosphorylation.

Cyclic AMP‐Dependent Protein Phosphorylation in Isolated Neuronal Growth Cones from Developing Rat Forebrain

We have shown recently that neuronal growth cones isolated from developing rat forebrain possess an appreciable activity of adenylate cyclase, which produces cyclic AMP and can be stimulated by various neurotransmitter receptor agonists and by forskolin. To investigate cyclic AMP-mediated biochemical mechanisms in isolated growth cones, we have centered the present study on cyclic AMP-dependent protein phosphorylation. One-dimensional gel electrophoretic analysis showed that cyclic AMP analogs increased incorporation of 32P into several phosphoproteins in molecular mass ranges of 50-58 and 76-82 kilodaltons, including those of 82, 76, and 51 kilodaltons. Two-dimensional electrophoresis, using isoelectric focusing in the first dimension, resolved phosphorylated alpha- and beta-tubulin species, actin, a very acidic protein (isoelectric point 4.0) with a molecular mass of 93 kilodaltons, and two proteins (x and x') closely neighboring beta-tubulin. Two other phosphoproteins seen in the gels had molecular masses of 56 and 51 kilodaltons (respective isoelectric points, 4.5 and 4.4) and, along with the 93-kilodalton phosphoprotein, were highly enriched in the isolated growth cones. Only the tubulin and actin species were major proteins in the isolated growth cones. Cyclic AMP analogs enhanced incorporation of 32P into phosphoproteins x and x', and, as assessed by immunoprecipitation, into beta-tubulin. Peptide digest experiments suggested that phosphoproteins x and x' are unrelated to beta-tubulin. Nonequilibrium two-dimensional electrophoresis resolved many phosphoproteins, of which a 79- and 75-kilodalton doublet, a 74-kilodalton species, and a 58-kilodalton doublet showed enhanced incorporation of 32P in the presence of cyclic AMP.

Cysteine proteinase inhibitors cause the accumulation of ubiquitin-polypeptide conjugates in lysosomes of 3T3-L1 fibroblasts

of myosin light chain phosphorylation (e.g. England, 1983). In contrast, we have observed an increase in the phosphorylation of myosin light chain in response to both SK&F 94 120 and isoprenaline. Interestingly, it appeared that SK&F 94 120 caused a relatively greater phosphorylation of myosin light chain. These data are consistent with SK&F 94120 exerting its inotropic effects in guinea-pig hcart by inhibition of phosphodiesterase, and the subsequent increase in the levels of cyclic AMP-dependent protein phosphorylation. Although qualitatively the phosphorylation patterns were similar for both SK&F 94 120 and isoprenaline. quantitative differences were observed, particularly in the phosphorylation of myosin light chain. England, P. J . (1976) Hiochrm. J . 160 .295304 England, P. J . ( 198.7) in Cordicrc Metdwlisrn (Drake-Holland, A. J.