Presence Of Cyclic AMP Research Articles

Cyclic AMP regulates expression of the rat gene for steroid 17α-hydroxylase / C 17–20 lyase P−450 (CYP17) in rat Leydig cells

The upstream region of the rat CYP17 gene shows significant homology to the upstream regions of the bovine and human genes, 53 and 60 percent, respectively. The start site of transcription was determined by primer extension and S1 nuclease protection to be 41 base pairs (bp) upstream of the initiating methionine codon. Expression vectors were constructed by ligation of upstream sequences into promoterless chloramphenicol acetyl transferase (CAT) vectors. Transient transfection studies using primary cultures of rat Leydig cells indicate a strong cAMP-responsive element located within the -26/+ 65 region. Stimulation by cyclic AMP was abolished when sequences upstream of -264 were included in expression vectors. No significant expression was seen in Leydig cells in the absence of dbcAMP nor was there any expression in the presence or absence of dbcAMP in rat skin fibroblasts or in mouse adrenal (Y-1) cells in which CYP17 is not normally expressed. Three possible regulatory elements were found in the 5′ upstream region: a CRE/ATF consensus sequence (GACGTCA) starting at position -57; a GRE consensus sequence (TGTTCT) starting at position -501; and a consensus sequence for AP-1 binding (TTAGTCA) starting at position -659. It was concluded that the CRE/ATF at -57 is not responsible for increased transcription in the presence of cyclic AMP.

Morphogenesis and differentiation of Dictyostelium cells interacting with immobilized glucosides: dependence on DIF production

Abstract Previous work has shown that multicellular morphogenesis of submerged Dictyostelium cells is inhibited when they bind to glucosides covalently linked to polyacrylamide gels. The amoebae aggregate normally, but then the aggregates repeatedly disperse and reaggregate, whereas control cells go on to form tight aggregates. We have investigated the role of the stalk cell differentiation inducing factors (DIFs) in this process. In the presence of cyclic AMP, amoebae submerged at high cell density accumulate DIP and differentiate into stalk cells. We find that stalk cell differentiation is inhibited by interaction of the cells with glucoside gels in these conditions, but can be restored by the addition of exogenous DIF–1. Since the responsiveness of cells to DIF–1 is not altered, it appears likely that the effect of the glucoside gel is to block DIF–1 production. Further, the addition of DIF–1 or DIF–2 stimulates the formation of tight aggregates by cells developing on glucoside gels in the absence of cyclic AMP, thus preventing the rounds of aggregation and disaggregation otherwise seen. This suggests a role for DIF in morphogenesis as well as in controlling cell differentiation. We propose a model in which immobilized glucosides activate a specific receptor (‘food sensor’) which drives the amoebae toward the vegetative state and inhibits DIF accumulation. DIF, on the other hand, induces tight aggregate formation and so locks the amoebae into the developmental program.

Phosphorylation of Nuclear Proteins in Myelinating Oligodendrocytes and Its Control by Cyclic AMP

Oligodendroglial nuclei isolated from rat brains at different stages of myelinogenesis (10, 18, and 30 days of age) were incubated with [gamma-32P]ATP and extracted with 0.75 M perchloric acid to yield a fraction of nonacidic chromatin proteins. The protein extracts were then analyzed by polyacrylamide gel electrophoresis. The phosphorylation pattern of these proteins was found to be different for different age groups. In 10-day-old rat oligodendrocytes the most extensive phosphorylation occurred in low molecular mass species (less than 30 kDa), in contrast to fractions obtained from 18- and 30-day-old rat oligodendrocytes which showed a significantly higher labeling of the proteins with molecular masses greater than 30 kDa. The phosphorylation of the latter species was greatly stimulated by the presence of cyclic AMP in the incubation media. The results suggest that the phosphorylation of specific nuclear proteins, which may play a regulatory role at different stages of oligodendroglial maturation and myelinogenesis, may be at least partially modulated by intracellular cyclic AMP.

Cellular HSP90 (HSP86) mRNA level and in vitro differentiation of mouse embryonal carcinoma (F9) cells

Treatment of mouse embryonal carcinoma (F9) cells with retinoic acid, an inducer of F9 cell differentiation, greatly increased the level of mRNA specific to one of the heat-shock proteins (HSP86). Experiments including the one employing differentiation-resistant mutant F9 cells suggested that the increase represents early molecular events associated with the embryonal differentiation. The increased HSP86 mRNA declined to the original level during further incubation. The presence of cyclic AMP, which stimulates conversion of the retinoic acid-induced primitive endoderm cells to parietal endoderm cells, prevented the decline. These results suggest that not only the elevation of HSP86 mRNA level represents early molecular events in F9 cell differentiation but also that sustaining the elevated level (by cyclic AMP) is associated with further differentiation of the embryonal cells.

Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes

Bioluminescent marine bacteria of the species Vibrio fischeri are the specific light organ symbionts of the sepiolid squid Euprymna scolopes. Although they share morphological and physiological characteristics with other strains of V. fischeri, when cultured away from the light organ association the E. scolopes symbionts depress their maximal luminescence over 1,000-fold. The primary cause of this reduced luminescence is the underproduction by these bacteria of luciferase autoinducer, a molecule involved in the positive transcriptional regulation of the V. fischeri lux operon. Such an absence of visible light production outside of the symbiotic association has not been previously reported among light organ symbionts of this or any other species of luminous bacteria. Levels of luminescence approaching those of the E. scolopes bacteria in the intact association can be restored by the addition of exogenous autoinducer to bacteria in laboratory culture and are affected by the presence of cyclic AMP. We conclude that some condition(s) specific to the internal environment of the light organ is necessary for maximal autoinduction of luminescence in the symbionts of this squid-bacterial association.

Partial characterization of cyclic AMP-dependent protein kinases in guinea-pig lung employing the synthetic heptapeptide substrate, Kemptide: In vitro sensitivity of the soluble enzyme to isoprenaline, forskolin, methacholine and leukotriene D 4

This paper describes the partial characterization of soluble cyclic AMP-dependent protein kinase (A-kinase) in guinea-pig lung using Kemptide, a synthetic serine-containing heptapeptide, and examines the sensitivity of this enzyme to drugs which are reported to increase and to decrease the intracellular concentration of cyclic AMP. Differential centrifugation of lung homogenates revealed that 78% of A-kinase was present in the 31,000 g max × 15 min supernatant fraction. Both basal and cyclic AMP-stimulated phosphotransferase activity of this ‘soluble’ enzyme were abolished by the heat-stable inhibitor of A-kinase. Soluble A-kinase was Mg 2+-dependent (apparent K m and K act 8.6 and 2.6 mM, respectively) and was stimulated nine-fold by saturating concentrations of both cyclic AMP ( K act: 131 nM) and cyclic GMP ( K act: 28.7 μM) at a protein (enzyme) concentration of 1.3 μg. Kinetic analysis of the effect of Kemptide and ATP revealed linear, Hanes plots with Michealis constants of ca. 12 and 13 μM, respectively. Chromatography of the soluble enzyme over DEAE-cellulose resolved three peaks of catalytic activity when fractions were assayed in the presence of cyclic AMP (10 μM): (i) free catalytic subunits (5%), (ii) Type I isoenzyme (5%) and (iii) Type II isoenzyme (90%). The A-kinase activity ratio was markedly increased in lung pre-treated with the smooth muscle relaxants isoprenaline and forskolin. This biochemical effect was both time- and concentration-dependent and was temporally associated with the ability of these drugs to reduce lung parenchymal tone. In contrast, the contractile agonists, methacholine (Mch) and leukotriene (LT) D 4 exerted opposite effects on A-kinase activity. Thus, Mch significantly reduced cyclic AMP levels and lowered basal A-kinase activity whilst the converse was true for LTD 4. For both drugs this biochemical effect accompanied contraction of the lung. Pre-treatment of lung tissue with flurbiprofen, an irreversible inhibitor of cyclo-oxygenase in vitro, abolished the ability of LTD 4 to increase the A-kinase activity ratio suggesting that this biochemical response was mediated indirectly through the stimulated biosynthesis and release of a prostanoid(s) able to activate adenylyl cyclase; the increase in tension induced by LTD 4, however, was not significantly affected by flurbiprofen pre-treatment. Collectively, these data support the concept that soluble A-kinase activity in guinea-pig lung can be regulated by changes in intracellular cyclic AMP and that activation and/or inhibition of this biochemical cascade may influence alterations in lung contractility. The ability of Mch to reduce A-kinase activity suggests that protein dephosphorylation may represent a novel mechanism by which agonists that interact with muscarinic cholinoceptors control or regulate cell function.

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

Induction of ketogenesis and fatty acid oxidation by glucagon and cyclic AMP in cultured hepatocytes from rabbit fetuses. Evidence for a decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition after glucagon or cyclic AMP treatment

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.

Phosphorylation of superior cervical ganglion proteins during regeneration.

The incorporation of radioactive phosphate into proteins of both normal and regenerating ganglia of the sympathetic nervous system of the rat is reported. The incorporation reactions were carried out in vitro by incubating homogenates of excised ganglia with [gamma-32P]ATP under various conditions. It was found that incorporation of phosphate into proteins of regenerating ganglia in the molecular mass range 10,000-100,000 daltons increased up to 40% over incorporation into proteins from control ganglia during the first 3 days following injury and returned to control levels after 14 days. Analysis of the proteins by two-dimensional electrophoresis revealed that only few, i.e., less than 20, became radioactively labelled in homogenates of superior cervical ganglia in the presence of Ca2+, and even fewer in the presence of cyclic AMP. Furthermore, all these proteins fell within a narrow pI range of 4-6. The growth-associated protein, variously designated GAP-43, B-50, F-1, and pp46, has an enhanced level of expression and phosphorylation in regenerating ganglia compared with controls at day 3. Injury also caused consistently higher levels of incorporation into two other proteins with molecular masses at positions 55,000 and 85,000 and pI values of 5.1 and 4.5, respectively; the former protein most probably is beta-tubulin. The fact that both proteins are found in the 15,000 g pellet after the tissue has been solubilized in 0.5% nonionic detergent indicates that they may indeed by components of filament assemblies. Thus, the results suggest that protein phosphorylation is a mechanism involved in cytoskeletal function in regenerating nerve.

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.

MRNA decay rates in late-developing Dictyostelium discoideum cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell-type-specific mRNAs.

We reevaluated the use of 32PO4 pulse-chases for analyzing mRNA decay rates in late-developing Dictyostelium cells. We found that completely effective PO4 chases could not be obtained in developing cells and that, as a consequence, the decay rates exhibited by some mRNAs were influenced by the rates at which they were transcribed. In developing cells disaggregated in the presence of cyclic AMP, the poly(A)+ mRNA population turned over with an apparent half-life of 4 h, individual mRNA decay rates were heterogeneous, and some prestalk and prespore mRNAs appeared to decay with biphasic kinetics. In cells disaggregated in the absence of cyclic AMP, all prestalk and prespore mRNAs decayed with biphasic kinetics. During the first 1 to 1.5 h after disaggregation in the absence of cyclic AMP, the cell-type-specific mRNAs were selectively degraded, decaying with half-lives of 20 to 30 min; thereafter, the residual prestalk and prespore mRNA molecules decayed at rates that were similar to those measured in the presence of cyclic AMP. This short-term labilization of cell-type-specific mRNAs was observed even for those species not requiring cyclic AMP for their accumulation in developing cells. The observation that cell-type specific mRNAs can decay at similar rates in disaggregated cells with or without cyclic AMP indicates that this compound does not act directly to stabilize prestalk and prespore mRNAs during development and that its primary role in the maintenance of cyclic-AMP-dependent mRNAs is likely to be transcriptional.

MRNA decay rates in late-developing Dictyostelium discoideum cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell-type-specific mRNAs.

We reevaluated the use of 32PO4 pulse-chases for analyzing mRNA decay rates in late-developing Dictyostelium cells. We found that completely effective PO4 chases could not be obtained in developing cells and that, as a consequence, the decay rates exhibited by some mRNAs were influenced by the rates at which they were transcribed. In developing cells disaggregated in the presence of cyclic AMP, the poly(A)+ mRNA population turned over with an apparent half-life of 4 h, individual mRNA decay rates were heterogeneous, and some prestalk and prespore mRNAs appeared to decay with biphasic kinetics. In cells disaggregated in the absence of cyclic AMP, all prestalk and prespore mRNAs decayed with biphasic kinetics. During the first 1 to 1.5 h after disaggregation in the absence of cyclic AMP, the cell-type-specific mRNAs were selectively degraded, decaying with half-lives of 20 to 30 min; thereafter, the residual prestalk and prespore mRNA molecules decayed at rates that were similar to those measured in the presence of cyclic AMP. This short-term labilization of cell-type-specific mRNAs was observed even for those species not requiring cyclic AMP for their accumulation in developing cells. The observation that cell-type specific mRNAs can decay at similar rates in disaggregated cells with or without cyclic AMP indicates that this compound does not act directly to stabilize prestalk and prespore mRNAs during development and that its primary role in the maintenance of cyclic-AMP-dependent mRNAs is likely to be transcriptional.

Cloning of the fic-1 gene involved in cell filamentation induced by cyclic AMP and construction of a delta fic Escherichia coli strain.

PA3092 is an Escherichia coli mutant that forms filaments at 43 degrees C in the presence of cyclic AMP (cAMP). The mutation responsible for this phenotype is called fic-1. We cloned fic-1 from PA3092 by selection for the neighboring argD gene. The fic-1 gene product had a relative molecular mass of 21 kilodaltons by the maxicell method. A strain with the fic gene completely deleted was constructed by replacing fic with a kanamycin resistance gene. In one of the fic-deleted strains derived from PA3092, cAMP did not induce cell filamentation at 43 degrees C, but it did in the same strain harboring a plasmid containing the fic-1 gene. These results indicate that the fic-1 gene product is necessary for the induction of cell filamentation by cAMP but is dispensable to the cell. We also found that high levels of NaCl suppressed the cell filamentation induced by cAMP.

The requirement for DIF for prestalk and stalk cell formation in Dictyostelium discoideum: A comparison of in vivo and in vitro differentiation conditions

In Dictyostelium discoideum stalk cell formation is induced by cyclic AMP and differentiation-inducing factor (DIF) when cells are plated in in vitro monolayers (Kay et al., 1979, Differentiation 13: 7–14). The in vivo developmental stages at which cells became independent of these factors were determined. Independence was defined as the stage at which dispersed cells no longer required the factors for stalk cell formation in low density monolayers. Cyclic AMP independent cells were first detected at around 12 hr of development, a time that corresponds to the transition between the tipped aggregate and the first finger stages. In contrast cells did not become independent of DIF until late culmination. The prestalk cell-specific isozyme acid phosphatase II and a stalk cell-specific 41,000 M r antigen (ST 41) were expressed during differentiation in low density monolayers in the presence of both cyclic AMP and DIF, but neither component was expressed in the presence of cyclic AMP alone. This result implies that DIF is essential for both prestalk and stalk cell formation. The two components were expressed within 2 hr of each other during differentiation in vitro, whereas during development in vivo acid phosphatase II was first detected at the first finger stage and ST 41 was first detected during late culmination, 8–12 hr later. These contrasting results suggest that the conversion of prestalk cells to stalk cells is unrestrained in monolayers, following directly after prestalk cell induction, but restrained in vivo until the culmination stage. This interpretation is consistent with the finding that cells become independent of DIF early during in vitro differentiation ( A. Sobolewski, N. Neave, and G. Weeks, 1983, Differentiation 25, 93–100), but do not become independent of DIF until the culmination stage when differentiating in vivo.

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

Mutants of the catabolite activator protein of Escherichia coli that are specifically deficient in the gene-activation function.

In the presence of cyclic AMP, the catabolite activator protein (CAP) of Escherichia coli binds DNA and stimulates transcription at a number of promoters. We have examined a model of CAP bound at the gal promoter and, using directed mutagenesis, have isolated CAP mutants that are analogous to the lambda repressor positive control (pc) mutants. These CAP mutants bind DNA but are defective in stimulating transcription at the gal P1 promoter. These mutants are also altered in positive control at the lac and malT promoters, where CAP binds to sites further upstream from the transcription start site.

Protein phosphorylation in yeast mitochondria: cAMP-dependence, submitochondrial localization and substrates of mitochondrial protein kinases.

We describe the identification and submitochondrial localization of four protein kinases and of their target proteins in derepressed yeast mitochondria. The activity of one of the kinases depends on the presence of cyclic AMP (cAMP). It is soluble and localized in the mitochondrial intermembrane space. Its natural target is a polypeptide of 40 kDa molecular mass, which is bound to the inner membrane. Besides this natural target this kinase phosphorylates acidic heterologous proteins, like casein, with high efficiency. The other protein kinases identified so far are cAMP-independent. At least one is localized in the matrix having its natural substrates (49 and 24 kDa) in the same compartment. Two others are firmly bound to the inner membrane phosphorylating target proteins in the inner membrane (52.5 kDa) and in the intermembrane space (17.5 kDa), respectively.

Activation of adenylate cyclase in human platelet membranes by guanosine 5'-[beta gamma-imido]triphosphate is inhibited by cyclic-AMP-dependent phosphorylation. Slow activation occurs in the absence of ATP.

Incubation of platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate causes a slow increase in GS (stimulatory GTP-binding protein) activation of adenylate cyclase. Mg2+ is necessary for this slow activation. This process is inhibited in the presence of ATP, and inhibition is greater if cyclic AMP is also included in the incubation. Adenosine 5'-[beta gamma-imido]triphosphate instead of ATP in the incubation facilitates the slow activation in the presence of cyclic AMP, and incubation of membranes with cyclic-AMP-dependent protein kinase inhibitor decreased inhibition of the slow activation of adenylate cyclase by ATP and cyclic AMP. A protein of 45 kDa in platelet membranes is phosphorylated in a cyclic-AMP-dependent manner. The transition from a reversibly activated form of GS to an irreversibly activated form is substantially slower in the presence of ATP and cyclic AMP. We propose that guanosine 5'-[beta gamma-imido]triphosphate-activated GS may exist in phosphorylated or non-phosphorylated forms, and that the non-phosphorylated form is the more active of the two species. The non-phosphorylated form of GS may correspond to the irreversibly activated state.

Activation of tyrosine hydroxylase in PC12 cells by the cyclic GMP and cyclic AMP second messenger systems.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.

Protein kinase activities associated with ribosomes of developing rat brain. Identification of eukaryotic initiation factor 2 kinases

Protein kinases associated with ribosomes in the brains of suckling (4–10 days) and adult (2 months) rats were extracted from ribosomal fraction with 0.5 M KCl. The different protein kinase activities were characterized by their ability to phosphorylate three exogenous substrates: casein, histone IIs and histone IIIs in the presence of different modulators. Ribosomal salt wash fractions contain a high casein kinase activity which was partially inhibited by heparin and stimulated by calmodulin in the presence of Ca2+, indicating the presence of casein kinase I and II and calcium/calmodulindependent kinases. Cyclic AMP and cyclic GMP-dependent kinases and protein kinase C (calcium/phospholipids-dependent kinase) were also present. No differences were found in the casein kinase activities of suckling and adult animals, but histone kinase activities were higher in adult than in suckling animals.To identify initiation factor 2 kinases, purified factor from adult brains was used as a protein marker. In addition to the phosphorylation of both factor subunits α and β by casein kinase I or II, an increased phosphorylation was detected of a subunit in the presence of cyclic AMP, and β subunit, in the presence of Ca2+/calmodulin or Ca2+/phospholipids.Present results reinforce our hypothesis that, as occurs in other eukaryotic cells, the decreased rate of protein synthesis during brain development may be regulated by phosphorylation of initiation factor 2.