Identification and characterization of a 3',5'-cyclic adenosine monophosphate-responsive element in the human corticotropin-releasing hormone gene promoter.

The regulation of human corticotropin-releasing hormone (hCRH) gene promoter activity by inducers of cAMP was investigated by transient transfection with a construct containing the hCRH gene promoter fused to the chloramphenicol acetyltransferase gene. Expression of hCRH-chloramphenicol acetyltransferase was strongly enhanced by forskolin in the neuroblastoma SK-N-MC and choriocarcinoma JAR cell lines. Overexpression of the catalytic subunit of protein kinase A dispensed the need for forskolin, and cotransfection of cAMP-responsive element-binding protein cDNAs enhanced forskolin-dependent expression of the hCRH promoter. Progressive 5'-end deletions of the hCRH promoter delineated a cAMP- responsive region between -226 and -164 base pairs. This fragment contained the sequence TGACGTCA at -221 base pairs, consistent with the consensus motif for a CRE. A homologous oligonucleotide responded to cAMP when cloned in either orientation in front of the thymidine kinase promoter. However, the level of constitutive and inductive cAMP expression was dependent on the cell line and on intrinsic properties of the promoter. Mutation of the wild type CRH-CRE sequence into an AP-1 site (TGAGTCA) completely abolished stimulation by cAMP. In contrast, coexpression of the catalytic subunit of protein kinase A dispensed the need for stimulation with forskolin, which showed that the CRH-CRE oligonucleotide served as a functional equivalent of the native CRE element.     

Characterization of a fluorescent substrate for the adenosine 3',5'-cyclic monophosphate-dependent protein kinase

A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation.     

Autophosphorylation of adenosine 3',5'-monophosphate-dependent protein kinase from bovine brain

A highly purified adenosine 3′,5′-monophosphate-dependent protein kinase from bovine brain has been found to catalyze its own phosphorylation. The incorporated phosphate was shown to be associated with the cyclic AMP-binding subunit (R-protein) of the protein kinase. The catalytic subunit exhibited no detectable incorporation of phosphate into itself, but was required for the phosphorylation of R-protein. The molecular weight of R-protein was determined by polyacrylamide gel electrophoresis to be about 48,000 in the presence of sodium dodecyl sulfate. Cyclic AMP strikingly inhibited the rate of autophosphorylation observed in the presence of ZnCl₂, CaCl₂, NiCl₂, or FeCl₂, but had no significant effect in the presence of MgCl₂ or CoCl₂. The concentration of cyclic AMP required to give half-maximal inhibition of phosphorylation was 3 × 10^?7m in the presence of either CaCl₂ or ZnCl₂. Guanosine 3′,5′-monophosphate was far less effective under the same experimental conditions than cyclic AMP. R-protein appears to be similar to a phosphoprotein recently discovered in synaptic membrane fractions from rat and bovine cerebral cortex.     

Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase.

The events involved in platelet shape change, aggregation, the release reaction and contraction are thought to be mediated by the availability of Ca2+. Increased cytoplasmic calcium, released from intracellular stores, triggers platelet activity, and increased concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits platelet alterations. We have studied the hypothesis that cyclic AMP may regulate the level of platelet cytoplasmic calcium by stimulating calcium removal by a membrane system. Such a hypothesis would be consistent with the reversibility of most manifestations of platelet activation. Human platelets were sonicated and unlysed platelets, mitochondria and granules were removed by centrifugation at 19 000 X g. Electron microscopy shows that the sediment, after centrifugation of the supernatant at 40 000 X g consists to a large extent of membrane vesicles. Such preparations actively concentrate calcium, as measured by the uptake of 45Ca, and also have the maximal calcium-stimulated ATPase activity. Optimal calcium uptake requires ATP and oxalate, and release of calcium from loaded vesicles was stimulated by the calcium ionophore A23187 and inhibited by LaCl3. These data indicate that calcium was being actively concentrated within membrane vesicles. After washing of such preparations in the absence of ATP, their capacity to take up Ca2+ is reduced to an initial value of 2.8 nmol/mg protein per min. In the presence of 2 - 10(6) M cyclic AMP to which was added a protein kinase preparation from human platelets, up to a 3-fold increase of this rate of uptake was observed. These results suggest that in platelets, as in muscle, cyclic AMP is a regulatory factor in the control of cytoplasmic calcium. Although the cyclic nucleotide may have still other functions, it appears likely that the well-known inhibition of many platelet activities by high intracellular cyclic AMP concentrations is directly linked to the stimulation of the removal of Ca2+ from the cytoplasm.     

Investigation of the adenosine 3', 5'-cyclic phosphate binding site of pig brain histone kinase with the aid of some analogues of adenosine 3', 5'-cyclic phosphate.

2'-O-Chloroacetyl cyclic AMP, 2'-O-acrylyl cyclic AMP and N-6, 2'-O-diacrylyl cyclic AMP were synthesized by the reaction of cyclic AMP with chloroacetic and acrylic anhydrides, respectively. Selective O-deacylation of N-6, 2'-O-diacrylyl cyclic AMP yielded N-6 -monoacrylyl cyclic AMP. In the reaction of gamma-mercaptobutyric acid with 8-bromo cyclic AMP, 8-(gamma-carboxypropylthio) cyclic AMP was obtained. The compounds synthesized and other cyclic AMP analogues (8-bromo cyclic AMP and adenosine 3', 5'-cyclic sulphate) were tested for ability to interact with the highly purified pig brain histone kinase. All compounds under study were found to be activators of the enzyme. The highest activating potency was manifested by 8-bromo cyclic AMP and 8-(gamma-carboxypropylthio) cyclic AMP; adenosine 3', 5'-cyclic sulphate was the least potent in this respect. All compounds were shown to inhibit binding of cyclic [-3-H]AMP to histone kinase. The inhibition was competitive with respect to cyclic AMP in all cases. All compounds, except for 2'-O-chloroacetyl cyclic AMP may indicate the formation of a covalent bond between this analogue and the enzyme. These findings suggest that an active site of the regulatory subunit of the histone kinase contains at least three specific areas responsible for cyclic AMP binding.     

Activation of 3',5'-cyclic adenosine monophosphate phosphodiesterase by calcium ion and a protein activator.

3',5'-CAMP phosphodiesterase was partially purified from bovine cerebral cortex. A heat-stable activating factor was separated from the enzyme by chromatography on DEAE-cellulose. The enzyme in crude ammonium sulfate fractions was stimulated by 5 mM CaCl2. This stimulation was reversed by the calcium chelator EGTA. The main phosphodiesterase peak obtained by DEAE-cellulose chromatography was not stimulated by Ca2+. Upon addition of column effluent containing a heat stable factor, Ca2+ activation was restored. Protein activator was inactive when endogenous contaminating Ca2+ was complexed with EGTA. It was concluded that activation of phosphodiesterase requires the presence of both activator and Ca1+. From an analysis of activation of cGMP hydrolysis a kinetic model for the interaction of Ca2+ and protein activator with the phosphodiesterase was developed. Heterotropic cooperativity between the binding of Ca2+ and protein activator to the phosphodiesterase was observed, i.e., Ca1+ decreased the apparent dissociation constant for protein activator and protein activator decreased the apparent dissociation constant for Ca2+.     

Further studies on the properties of the rabbit reticulocyte adenosine 3',5'-cyclic monophosphate-dependent protein kinase I

The properties of cyclic AMP-dependent protein kinase I isolated from rabbit reticulocytes were further investigated. The enzyme catalyzes the phosphorylation of histone in the presence of ATP and Mg²⁺ and this reaction is stimulated by cyclic AMP. The pH optimum of the reaction was between 8.5 and 9.0, when assayed in the presence of cyclic AMP. No distinct pH optimum was observed in the absence of the cyclic nucleotide. The K_m values for ATP appeared to be very similar whether it was determined in the presence (K_m = 1.7 × 10⁻⁴m) or absence (K_m = 2.5 × 10⁻⁴m) of cyclic AMP. The rate of heat inactivation of the catalytic activity and the cyclic AMP binding activity of kinase I were found to be dependent on the presence of Mg²⁺, ATP, and/or cyclic AMP. In the presence of cyclic AMP, the rate of inactivation of the catalytic activity of kinase I at 53 ° was accelerated. On the other hand, the cyclic AMP binding activity appeared to be protected from heat inactivation by the cyclic nucleotide. When both ATP and Mg²⁺ were present in the heating mixture, no loss of catalytic and binding activities of kinase I were observed even up to 8 min of heating at 53 °. The cyclic AMP binding activity of kinase I was almost completely inhibited by mercuric acetate at a concentration of 1 mm, while the loss in catalytic activity was only 50%. These results substantiate our previous observation that kinase I contains two nonidentical subunits, a catalytic subunit and a cyclic AMP binding subunit.     

A cyclic adenosine 3',5'-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa

An intracellular cAMP-PKA signaling plays a pivotal role in the expression of fertilizing ability in mammalian spermatozoa. The aim of this study is to disclose biological function of serine/threonine protein kinases that are activated by the action of the cAMP-PKA signaling in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) at 38.5 degrees C up to 180 min, and then they were used for biochemical analyses of PKCs by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The incubation of spermatozoa with cBiMPS gradually activated PKCs in the connecting piece. The activation of sperm PKCs was accompanied with changes of their electrophoretic mobility by the PKA-mediated serine/threonine phosphorylation. In coincidence with the PKC activation, the cBiMPS-incubated spermatozoa were capable of exhibiting hyperactivation of flagellar movement. Moreover, the cBiMPS-induced hyperactivation was dramatically suppressed by the addition of either of specific PKC inhibitors (Ro-32-0432 and bisindolylmaleimide I) to the sperm suspensions. On the other hand, experiments using a calcium-deficient medium showed that the cBiMPS-induced hyperactivation of flagellar movement and activation of PKCs required the extracellular calcium. Based on the obtained data, we have concluded that a cAMP-PKA signaling can induce activation of calcium-sensitive PKCs that is leading to the hyperactivation of flagellar movement in boar spermatozoa. Moreover, the cAMP may have a unique role as the up-regulator of PKCs during the expression of fertilizing ability in boar spermatozoa.