cAMP-dependent Activity Research Articles

IDENTIFICATION OF A CYCLIC AMP‐DEPENDENT PROTEIN KINASE IN THE DINOFLAGELLATE AMPHIDINIUM OPERCULATUM

Cyclic AMP is an important signaling molecule in both prokaryotes and eukaryotes. In eukaryotes, the action of cyclic AMP is mediated by cyclic AMP‐dependent protein kinase (PKA). Efforts to identify PKA in dinoflagellates have met with mixed success in previous studies. In Amphidinium operculatum, we have identified the presence of cAMP, and observed cell cycle inhibition by the cAMP pathway inhibitor, isobutyl methylxanthine, suggesting the presence of a eukaryotic cAMP pathway. However, crude lysates of A. operculatum failed to show in vitro kinase activity toward kemptide, a specific substrate of mammalian PKA. This may reflect the presence of unidentified low molecular weight inhibitors, because when cell lysates were fractionated by gel filtration chromatography, cAMP dependent kinase activity was identified. The molecular weight of this PKA was 66 kDa, much smaller than bovine PKA (250 kDa), but similar to that reported in the diatom Cylindrotheca (78 kDa). Current work is focusing on characterization of subunit structure of the PKA of A. operculatum, as compared with bovine PKA.

Phosphodiesterase activity in neutrophils from horses with chronic obstructive pulmonary disease

Neutrophils are recruited to the lungs of horses with chronic obstructive pulmonary disease (COPD) and exhibit increased activity after antigen challenge. Phosphodiesterase type4 (PDE4) inhibitors have been shown to attenuate human neutrophil activation. The aim of this study was to establish the PDE isoenzyme profile of equine neutrophils using isoenzyme selective inhibitors to determine if these compounds should be evaluated in horses with COPD. Total cAMP and cGMP dependent PDE activity was no different in neutrophils from normal (156.2±7.1 and 6.8±0.6 pmol/ min/ mg for cAMP and cGMP, respectively) and COPD susceptible horses (146.0±10.2 and 5.5±0.6 pmol/ min/ mg for cAMP and cGMP, respectively). The PDE4 inhibitors, CDP840 and rolipram, caused significant, concentration related and almost complete inhibition of PDE activity (IC 50 values=8.8±0.1×10 −9 and 7.3±0.2×10 −9 M for CDP840; 1.2±0.1×10 −6 and 1.1±0.1×10 −6 M for rolipram in normal and COPD susceptible horses, respectively). The inhibitory effects of the mixed PDE3/ PDE4 inhibitor, zardaverine were of similar magnitude and potency to rolipram. However, the limited inhibitory effects of the PDE3 inhibitor, siguazodan, suggest that zardaverine is acting primarily via PDE4 inhibition. These results indicate that PDE4 is the predominant isoenzyme present in the equine neutrophil and inhibition of PDE activity using selective PDE4 inhibitors may, therefore, modulate equine neutrophil activation in horses with COPD.

Identification and Characterization of Basal and Cyclic AMP Response Elements in the Promoter of the Rat GTP Cyclohydrolase I Gene

5812 base pairs of rat GTP cyclohydrolase I (GTPCH) 5'-flanking region were cloned and sequenced, and the transcription start site was determined for the gene in rat liver. Progressive deletion analysis using transient transfection assays of luciferase reporter constructs defined the core promoter as a highly conserved 142-base pair GC-rich sequence upstream from the cap site. DNase I footprint analysis of this region revealed (5' --> 3') a Sp1/GC box, a noncanonical cAMP-response element (CRE), a CCAAT-box, and an E-box. Transcription from the core promoter in PC12 but not C6 or Rat2 cells was enhanced by incubation with 8-bromo-cyclic AMP. Mutagenesis showed that both the CRE and CCAAT-box independently contribute to basal and cAMP-dependent activity. The combined CRE and CCAAT-box cassette was also found to enhance basal transcription and confer cAMP sensitivity on a heterologous minimal promoter. The addition of the Sp1/GC box sequence to this minimal promoter construct inhibited basal transcription without affecting the cAMP response. EMSA showed that nuclear proteins from PC12 but not C6 or Rat2 cells bind the CRE as a complex containing activating transcription factor (ATF)-4 and CCAAT enhancer-binding protein beta, while both PC12 and C6 cell nuclear extracts were recruited by the CCAAT-box as a complex containing nuclear factor Y. Overexpression of ATF-4 in PC12 cells was found to transactivate the GTPCH promoter response to cAMP. These studies suggest that the elements required for cell type-specific cAMP-dependent enhancement of gene transcription are located along the GTPCH core promoter and include the CRE and adjacent CCAAT-box and the proteins ATF-4, CCAAT enhancer-binding protein beta, and nuclear factor Y.

Involvement of cAMP and protein kinase A in conidial differentiation by Erysiphe graminis f. sp. hordei.

Erysiphe graminis f. sp. hordei, the causal agent of barley powdery mildew, is an obligate biotroph. On arrival on the host, a primary germ tube (PGT) emerges from the conidium. An appressorial germ tube (AGT) then appears, forms an appressorium, and effects host penetration. Such developmental precision may be due to multiple, plant-derived signals and to endogenous tactile and chemical signals. The transduction mechanism remains obscure. The isolation of an expressed sequence tag (EST) homologue of the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) enabled the corresponding gene to be characterized and the transcript to be identified in conidia and in PGT and AGT stage spores. cAMP-dependent PKA activity was detected in ungerminated conidia. These data suggest that PKA and cAMP are involved in conidial development. To substantiate this we exploited the responses of developing conidia to various surfaces, including exposure to the host leaf (fully inductive to AGT formation), cellulose membrane (semi-inductive), and glass (non-inductive). Assessment of fungal development, following application of exogenous cAMP or cAMP analogues, revealed that, at different concentrations and on different surfaces, cAMP either promoted or inhibited conidial differentiation. Various PKA inhibitors were tested for their effect on PKA activity and conidial development. A negative correlation was established between PKA inhibition in vitro and fungal development in vivo. Taken collectively, these data suggest that PKA and cAMP play a role in conidial differentiation in this obligate, plant-pathogenic fungus.

Modulation of ion channels: a "current" view of AKAPs.

Modulation of ion channels: a "current" view of AKAPs.

Activation of wild-type and deltaF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms.

The cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and its modulation through inhibition of phosphodiesterases (PDE) were studied with the cell-attached patch-clamp technique in Calu-3 cells (expressing endogenous CFTR) and NIH3T3 cells [expressing either wild-type (Wt)-CFTR or DeltaF508-CFTR]. In Calu-3 cells, CFTR current was augmented by increasing concentrations of 8-(4-chlorophenylthio)-adenosine 3', 5'-cyclic monophosphate (CPT-cAMP) and reached a saturating level at >/=60 microM. Varying the forskolin concentration also modulated CFTR activity; 10 microM was maximally effective since supplemental application of 200 microM CPT-cAMP had no additional effect. Activation of CFTR by increasing the cAMP concentration occurs through an increase of the NPo (product of the number of functional channels and the open probability) since the single-channel amplitude remains unchanged. In Calu-3 and NIH3T3-Wt cells, PDE inhibitors, milrinone (100 microM), 8-cyclopentyl-1, 3-dipropylxanthine (CPX, 25 microM), and 3-isobutyl-1-methylxanthine (IBMX, 200 microM), did not enhance CFTR current initially activated with 10 microM forskolin, but each potentiated CFTR activity elicited with a submaximal forskolin concentration (e.g., 100 nM) and prolonged the deactivation of CFTR channel current upon removal of forskolin. Millimolar IBMX increased the NPo of both Wt- and DeltaF508-CFTR even under maximal cAMP stimulation. Quantitatively, these effects of millimolar IBMX on NPo approximate those of genistein, which potentiates the cAMP-dependent CFTR activity via a mechanism that does not involve increases in cellular cAMP. Thus, depending on the concentration, PDE inhibitors may affect CFTR through different mechanisms.

Biglycan Gene Expression in the Human Leiomyosarcoma Cell Line SK-UT-1: BASAL AND PROTEIN KINASE A-INDUCED TRANSCRIPTION INVOLVES BINDING OF Sp1-LIKE/Sp3 PROTEINS IN THE PROXIMAL PROMOTER REGION

In this study we demonstrate that the gene encoding the small leucine-rich proteoglycan biglycan is expressed in human myometrial tissue and in the human leiomyosarcoma cell line SK-UT-1. Treatment of SK-UT-1 cells with forskolin or 8-bromo-cAMP strongly increased biglycan mRNA and this effect was transcriptional as shown by transient transfection experiments with biglycan promoter-luciferase reporter fusion genes. The cAMP-mediated induction of the transfected biglycan promoter in SK-UT-1 cells was abolished by coexpression of a specific protein kinase A inhibitor, and was mimicked by overexpression of the catalytic subunit (Cbeta) of protein kinase A. By 5' deletion analysis, part of the cAMP response was localized to the segment from residues -78 to -46 of the biglycan promoter. This region conferred strong cAMP responsiveness to a heterologous promoter. Electrophoretic mobility shift and antibody supershift assays identified two specific complexes that contained nuclear proteins antigenically related to the ubiquitous transcription factors Sp1 and Sp3, respectively. The binding site of these proteins was mapped to a CT-rich sequence extending from -59 to -49 in the biglycan promoter. Mutating this sequence eliminated complex formation and markedly reduced basal and cAMP-dependent promoter activity of transfected reporter genes. In vitro binding studies using recombinant Sp1 revealed that the nuclear factor binding to the CT element was not Sp1 but a Sp1-like protein(s). Western blot analysis of SK-UT-1 nuclear proteins confirmed expression of Sp3, Sp1 and nuclear proteins that crossreacted with Sp1 antibody but according to their molecular weight were not Sp1. These results indicate that all cAMP-dependent as well as some basal biglycan transcription in SK-UT-1 cells is mediated through activated protein kinase A and that both functions are conferred at the promoter level through the interaction of Sp1-like/Sp3 factors with the CT element at -59 in the biglycan promoter.

Cystic fibrosis transmembrane conductance regulator activation by cAMP-independent mechanisms.

Recent studies have demonstrated that several compounds with diverse structures can activate wild-type cystic fibrosis transmembrane conductance regulator (CFTR) by non-receptor-mediated mechanisms. Some of these compounds have been shown to enhance cAMP-dependent activation of DeltaF508-CFTR. This study was undertaken to compare the mechanisms by which genistein, IBMX, milrinone, 8-cyclopentyl-1, 3-dipropylxanthine (CPX), the benzimidazolone NS004, and calyculin A increase CFTR activity. Our studies demonstrate that, in transfected NIH-3T3 cells, maximal enhancements of forskolin-dependent DeltaF508-CFTR activity are greatest with genistein, IBMX, and NS004. Milrinone, genistein, CPX, NS004, and calyculin A do not increase cellular cAMP. Because forskolin and calyculin A increase in vivo phosphorylation of cAMP binding response element (CREB), the inability of milrinone, genistein, CPX, and NS004 to increase CREB phosphorylation suggests that they do not stimulate protein kinase A or inhibit phosphatase activity. Our data suggest that the mechanisms by which genistein and NS004 activate CFTR differ. We also demonstrate that, in NIH-3T3 cells, IBMX-dependent enhancement of cAMP-dependent CFTR activity is not due to an increase in cellular cAMP and may involve a mechanism like that of genistein.

CAMP-Mediated Regulation of Neurotrophin-Induced Collapse of Nerve Growth Cones

Neurotrophins are known to promote the survival, differentiation, and neurite outgrowth of developing neurons. Here we report that acutely applied brain-derived neurotrophic factor (BDNF) induces rapid growth cone collapse and neurite retraction of embryonic Xenopus spinal neurons in culture. The collapsing effect of BDNF depends on the activation of Trk receptor tyrosine kinase, requires an influx of extracellular Ca2+, and is regulated by cAMP-dependent activity. Elevation of intracellular cAMP levels ([cAMP]i) by forskolin or (Sp)-cAMP completely blocked the collapsing effect, whereas inhibition of protein kinase A (PKA) by (Rp)-cAMP potentiated the collapsing action. BDNF-induced growth cone collapse was only observed in 6 hr cultures but not in 24 hr cultures. However, inhibition of PKA by (Rp)-cAMP restored the collapsing response of these "old" neurons in 24 hr cultures, suggesting that embryonic Xenopus spinal neurons may upregulate their endogenous cAMP-dependent activity during development in culture, leading to the blockade of their collapsing response to BDNF. Taken together, our results suggest the presence of cross-talk between Ca2+- and cAMP-signaling pathways involved in the collapsing action of neurotrophins, in which the cAMP-pathway regulates the Ca2+-mediated signal transduction required for BDNF-induced collapse. By modulating the cAMP-dependent activity through the intrinsic programming or interaction with other factors present in the environment, a neuron thus could respond to the same extracellular factors with different morphological and cellular changes at different stages during development.

Different cis-acting elements are involved in the regulation of TRP1 and TRP2 promoter activities by cyclic AMP: pivotal role of M boxes (GTCATGTGCT) and of microphthalmia.

In melanocytes and in melanoma cells, cyclic AMP (cAMP)-elevating agents stimulate melanogenesis and increase the transcription of tyrosinase, the rate-limiting enzyme in melanin synthesis. However, two other enzymes, tyrosinase-related protein 1 (TRP1) and TRP2, are required for a normal melanization process leading to eumelanin synthesis. In B16 melanoma cells, we demonstrated that stimulation of melanogenesis by cAMP-elevating agents results in an increase in tyrosinase, TRP1, and TRP2 expression. cAMP, through a cAMP-dependent protein kinase pathway, stimulates TRP1 and TRP2 promoter activities in both B16 mouse melanoma cells and normal human melanocytes. Regulation of the TRP1 and TRP2 promoters by cAMP involves a M box and an E box. Further, a classical cAMP response element-like motif participates in the cAMP responsiveness of the TRP2 promoter, demonstrating that the TRP2 gene is subjected to different regulatory processes, which could account for its different expression patterns during embryonic development or under specific physiological and pathological conditions. We also found that microphthalmia, a basic helix-loop-helix transcription factor, strongly stimulates the transcriptional activities of the TRP1 and TRP2 promoters, mainly through binding to the M boxes. Additionally, we demonstrated that cAMP increases microphthalmia expression and thereby its binding to TRP1 and TRP2 M boxes. These convergent and compelling results disclose at least a part of the molecular mechanism involved in the regulation of melanogenic gene expression by cAMP and emphasize the pivotal role of microphthalmia in this process.

High Density Protein Kinase Assay With Subattomole Sensitivity

A rapid assay system based on incorporation of [γ-32P]ATP into biotinylated peptide substrates and their subsequent capture onto a high capacity streptavidin-coated membrane, SAM2™, has been developed for the detection of protein kinases. The system uses prenumbered and partially cut membrane squares for analyzing a limited number of samples or can be formatted to analyze up to 1,536 samples per microtiter plate footprint of 7.0 cm X 10.6 cm. The high biotin-binding capacity and low background of the membrane allows the use of nearly saturating amounts of most substrates, giving this system very high signal-to-noise ratios at low enzyme concentrations. Using cAMP-dependent Protein Kinase A (PKA) as a model system, as little as 0.3 amol of purified enzyme in 0.2 μl can be detected with a linear response range of over 3 orders of magnitude. cAMP-dependent kinase activity can be measured directly in tissue extracts by using a specific substrate and harsh washing procedures to reduce nonspecific backgrounds from proteins phosphorylated by other kinases. For increased assay flexibility, results can be analyzed either by PhosphorImager™ quantitation or by scintillation counting.

Anoxic injury of endothelial cells causes divergent changes in protein kinase C and protein kinase A signaling pathways.

Alterations in protein kinase C (PKC) and cAMP-dependent kinase have been documented in anoxic brain injury. However, the regulation of these signaling enzymes in the cerebrovasculature has not been explored. In this study, cultured brain endothelial cells exposed to anoxic injury (anoxia--20 min/reoxygenation--40 min) showed both a significant increase (p < 0.001) in PKC and decrease (p < 0.01) in cAMP-dependent kinase activity. Analysis of PKC by Western blot indicated an increase in kinase level in response to anoxic injury, whereas there was no change in the level of cAMP-dependent protein kinase, as measured by labeled cAMP binding. Inhibition of nitric oxide synthase did not affect these changes. Addition of the nitric oxide-releasing compound sodium nitroprusside caused a dose-dependent increase in the activity of both signaling systems in endothelial cells. These data demonstrate that anoxic injury of brain endothelial cells in culture causes significant and divergent changes in signaling kinase activity. Abnormalities in brain endothelial PKC and cAMP-dependent kinase could have important consequences for the blood-brain barrier in anoxic brain injury.

TTF-2 Does Not Appear to Be a Key Mediator of the Effect of Cyclic AMP on Thyroglobulin Gene Transcription in Primary Cultured Dog Thyrocytes

TTF-2 is a thyroid-specific winged-helix transcription factor which has been proposed to play a key role in the hormonal control of thyroglobulin and thyroperoxidase genes transcription in FRTL-5 cells. We have analyzed TTF-2 DNA-binding activity in primary cultures of dog thyrocytes maintained in control condition or in the presence of the cAMP agonist forskolin. Binding of35S-labelled nuclear proteins to the TTF-2 recognition sequence identified the presence of two molecular species of 41.5 and 42.5 kDa. TTF-2 DNA-binding activity was clearly detectable in nuclear extracts from unstimulated cells and appeared increased in forskolin-treated cells. Thus, the presence of TTF-2 DNA-binding activity does not correlate with the cAMP-dependent activity of thyroglobulin and thyroperoxidase genes in this cell system. In addition, the mutation of the TTF-2 binding site in the thyroglobulin promoter resulted in a very reduced but still clearly cAMP-dependent promoter activity when assayed by transient expression in the same cells. These results do not support a dominant role for TTF-2 in the cAMP-dependent control of thyroglobulin gene transcription in primary cultured thyrocytes.

CAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels.

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac alpha1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the alpha1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.

CAMP-induced switching in turning direction of nerve growth cones.

Development of the nervous system depends on the correct pathfinding and target recognition by the growing tip of an axon, the growth cone. Diffusible or substrate-bound molecules present in the environment may serve as either attractants or repellents to influence the direction of growth-cone extension. Here we report that differences in cyclic-AMP-dependent activity in a neuron may result in opposite turning of the growth cone in response to the same guidance cue. A gradient of brain-derived neurotrophic factor normally triggers an attractive turning response of the growth cone of Xenopus spinal neurons in culture, but the same gradient induces repulsive turning of these growth cones in the presence of a competitive analogue of cAMP or of a specific inhibitor of protein kinase A. This cAMP-dependent switch of the turning response was also found for turning induced by acetylcholine, but not for the turning induced by neurotrophin-3 (NT-3). Thus, in the presence of other factors that modulate neuronal cAMP-dependent activity, the same guidance cue may trigger opposite turning behaviours of the growth cone during its pathfinding in the nervous system.

Evidence that Functional Interactions of CREB and SF-1 Mediate Hormone Regulated Expression of the Aromatase Gene in Granulosa Cells and Constitutive Expression in R2C Cells

The proximal promoter of the rat aromatase CYP19 gene contains two functional domains that can confer hormone/cAMP inducibility in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Region A contains a hexameric sequence that binds steroidogenic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding factors X and Y had overlapping functions with CREB, and to determine if the CREB and SF-1 binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameric SF-1 binding site were generated. Mutations within the CRE showed that CREB but not factors X and Y mediated cAMP-dependent activity of chimeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the converse) resulted in a loss of approximately 50% cAMP-dependent CAT activity. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-dependent CAT activity. When these same constructs where transfected into R2C Leydig cells, mutation of either the CREB or SF-1 binding sites resulted in a greater than 90% loss of CAT activity. Western blot and immunocytochemistry analyses revealed that the amount of phosphorylated CREB increased in response to hormone/cAMP in granulosa cells and was high in R2C Leydig cells, coinciding with expression of the transgenes and endogenous aromatase mRNA in each cell type. Therefore, in both cell types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cells, whereas they interact synergistically to confer high basal transactivation in R2C Leydig cells. Taken together, the results indicated that the molecular mechanisms or pathways that activate CREB, SF-1 or their interaction are different in granulosa cells and R2C cells.

Evidence that functional interactions of CREB and SF-1 mediate hormone regulated expression of the aromatase gene in granulosa cells and constitutive expression in R2C cells

The proximal promoter of the rat aromatase CYP19 gene contains two functional domains that can confer hormone/cAMP inducibility in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Region A contains a hexameric sequence that binds steroidogenic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding factors X and Y had overlapping functions with CREB, and to determine if the CREB and SF-1 binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameric SF-1 binding site were generated. Mutations within the CRE showed that CREB but not factors X and Y mediated cAMP-dependent activity of chimeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the converse) resulted in a loss of approximately 50% cAMP-dependent CAT activity. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-dependent CAT activity. When these same constructs where transfected into R2C Leydig cells, mutation of either the CREB or SF-1 binding sites resulted in a greater than 90% loss of CAT activity. Western blot and immunocytochemistry analyses revealed that the amount of phosphorylated CREB increased in response to hormone/cAMP in granulosa cells and was high in R2C Leydig cells, coinciding with expression of the transgenes and endogenous aromatase mRNA in each cell type. Therefore, in both cell types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cells, whereas they interact synergistically to confer high basal transactivation in R2C Leydig cells. Taken together, the results indicated that the molecular mechanisms or pathways that activate CREB, SF-1 or their interaction are different in granulosa cells and R2C cells.

Functional interactions, phosphorylation, and levels of 3',5'-cyclic adenosine monophosphate-regulatory element binding protein and steroidogenic factor-1 mediate hormone-regulated and constitutive expression of aromatase in gonadal cells.

The proximal promoter of the rat aromatase CYP19 gene contains two functional regions that, by 5'-deletion analyses, have been shown to confer hormone/ cAMP inducibility to chimeric genes in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Promoter region A binds Steroidogenic Factor-1 (SF-1); region B binds cAMP-regulatory element binding protein (CREB) and two other factors (designated X and Y). Mutations were generated within the context of the intact promoter to selectively eliminate the binding of either SF-1, CREB, CREB plus factors X and Y, or all of the above. When expression vectors that failed to bind either CREB alone or CREB plus factors X and Y were transfected into granulosa cells, cAMP-dependent chloramphenicol acetyltransferase (CAT) activity was reduced 65% indicating that CREB alone, and not factors X and Y, mediates the cAMP response of this cAMP response element-like domain. Similarly, cAMP-dependent CAT activity was reduced 50% in constructs that failed to bind SF-1 and was abolished with vectors that were unable to bind either factor. In R2C Leydig cells, the absence of either CREB or SF-1 binding resulted in an almost complete loss in CAT activity. Both immunoreactive CREB and phosphorylated CREB (phospho-CREB) were present in extracts and nuclei of R2C cells. Immunoreactive phosphoCREB was low in granulosa cell extracts and nuclei but increased rapidly (90 min) in response to FSH/cAMP and was highest at 48 h, at a time when SF-1 was also phosphorylated and expression of the endogenous gene was elevated. Although the amount of CREB and SF-1 remained unchanged in response to FSH, LH mediated a rapid decrease in the amount of SF-1 (but not CREB) that is coincident with decreased aromatase mRNA in luteinizing granulosa cells. Taken together, the data indicate that expression of the aromatase gene is dependent on the additive interactions of regions A and B of the aromatase promoter in granulosa cells and the synergistic interactions of these same regions in R2C cells and that these interactions are dependent, in turn, on the phosphorylation of CREB and SF-1 and the content of these factors, as well as the presence of putative coregulatory molecules.

Cyclic-AMP-dependent activation of an inter-phylum hybrid histone-kinase complex reconstituted from sea urchin sperm-regulatory subunits and bovine heart catalytic subunits.

A cAMP-dependent histone kinase was purified and characterized from spermatozoa of the sea urchin Hemicentrotus pulcherrimus. The molecular mass of the kinase was estimated to be 178 kDa by native PAGE and 400 kDa by gel chromatography on a Superose 6 HR 10/30 column. The enzyme, composed of two 39-kDa catalytic subunits and two 48-kDa regulatory subunits, phosphorylates the lysine-rich histone subspecies (H1 and H2B) isolated from H. pulcherrimus spermatozoa. We isolated cDNA clones encoding a 39-kDa catalytic subunit and a 48-kDa regulatory subunit of the enzyme. The cDNA clone for the 39-kDa subunit was 3881 bp, and the 352-residue deduced amino acid sequence showed 78% similarity with the catalytic subunit of/mammalian cAMP-dependent protein kinase (PKA). The cDNA for the 48-kDa subunit was 4589 bp and the 368-residue deduced amino acid sequence showed 57% similarity with the regulatory subunit of mammalian PKA, although the N-terminal 77 residues showed poor similarity. The mRNAs encoding both the catalytic subunit (7.5 kb) and the regulatory subunit (4.6 kb) were expressed in testis, ovary and egg. An inter-phylum hybrid enzyme, reconstituted from the regulatory subunit of cAMP-dependent histone kinase of sea urchin sperm and the catalytic subunit of bovine heart PKA, has a cAMP-dependent histone kinase activity. Thus, we suggest that the N-terminal 77-amino-acid residues of the regulatory subunit are not essential for inhibition by the regulatory subunit of the catalytic subunit, and that cAMP-dependent inhibitory activity of the regulatory subunit resides in the sequence between the inhibitory site and the C-terminus.

Regulation of somatostatin gene transcription by cyclic adenosine monophosphate

Cyclic adenosine monophosphate (cAMP) stimulates transcription of somatostatin and other target genes with burstattenuation kinetics. The kinetics of protein kinase (PK-A)—dependent cAMP response element binding protein (CREB) phosphorylation closely parallel the changes in transcription of cAMP-responsive genes by run-on assay. Nuclear translocation of PK-A, visualized by microinjection of fluorescently labeled PK-A holoenzyme, appears to represent the rate-limiting step in CREB phosphorylation and transcriptional activation. We and others have recently characterized a CREB-binding protein (CBP), which specifically recognizes sequences within the Ser133 phosphorylated form of CREB. CBP does not regulate the DNA binding, dimerization, or nuclear targeting properties of CREB, but binds selectively to the kinase-inducible 60 amino acid trans-activation domain (KID) of CREB, critical for PK-A—inducible transcription. We developed an antiserum directed against amino acid 634–648 within the CREB-binding domain of CBP. We detected a 265-kd polypeptide by Western blot as predicted from the cDNA, which coincided with the predominant phospho-CREB—binding activity in Hela nuclear extracts by “Far Western” blot assay. An identical phospho-CREB—binding activity was also found in NIH-3T3 cells. This phospho-CREB—binding protein appeared to be specific for Ser133-phosphorylated CREB, because no such band was detected with CREB labeled to the same specific activity at a nonregulatory phosphoacceptor site (Ser156) by casein kinase II (CKII). Following microinjection into nuclei of NIH-3T3 cells, a cAMP response element (CRE)-lacZ reporter was markedly induced by treatment with 8-Br cAMP plus isobutyl methyl xanthine (IBMX). Coinjection of CBP antiserum with the CRE-lacZ plasmid inhibited cAMP-dependent activity in a dose-dependent manner, but control immunoglobulin G (IgG) had no effect on this response. We can now begin reconstituting PK-A—dependent transcription in vitro, using well-characterized proteins such as CREB, TAF 110, and CBP. The assembly of such factors on cAMP-regulated promoters like somatostatin may enable responsiveness to a variety of hormonal stimuli that employ cAMP as their second messenger.