Transcriptional suppression of the human T-cell leukemia virus type I long terminal repeat occurs by an unconventional interaction of a CREB factor with the R region.

To analyze regulation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR), cell lines were generated from LTR-tax x LTR-beta-galactosidase (beta-Gal) doubly transgenic mouse fibroblastic tumors. The HTLV-I LTR directs expression of both the tax and lacZ genes, and Tax up-modulates both promoters in primary cells. However, once cells were transformed by tax, beta-Gal but not tax expression was suppressed. Supertransformation of these cells with v-src suppressed both beta-Gal and tax expression. This suppression was reversed by treatment with the tyrosine kinase inhibitor herbimycin A or protein kinase A inhibitor H8. Electrophoretic mobility shift assays demonstrated augmented binding in the R but not U3 region. This binding was competitively inhibited by a high-affinity CREB oligodeoxynucleotide and super-shifted with a specific CREB antibody. Treatment of cells with the cyclic AMP analog dibutyryl cyclic AMP also transiently increased the R region binding dramatically. In vitro DNase I footprint analysis identified a protein-binding sequence in the R region which corresponded with suppression. However, this target sequence lacked a conventional CREB-binding site. A 70.5-kDa DNA-binding protein was partially purified by affinity chromatography, along with a 49-kDa protein which reacted with CREB-specific sera. These data demonstrate that HTLV-I LTR suppression is associated with CREB factor binding in the R region, probably by direct interaction with a 70.5-kDa protein, and provide a novel mechanism for maintenance of viral latency.     

Altered interaction between binding and catalytic subunits of a cyclic AMP-stimulated protein kinase from hepatoma cells.

Binding activity obtained from an established line of hepatoma tissue culture (HTC) cells has a lower apparent affinity for cyclic AMP at physiological pH than has the analogous binding activity from rat liver. However, the apparent binding affinity of HTC preparations can be reversibly increased by adding NaCl or guanidine · HCl. In the presence of such activating substances, a macromolecular inhibitory activity has been chromatographically separated from the cyclic AMP-binding activity. Removal of this inhibitory component causes the apparent affinity of the cyclic AMP-binding activity from HTC cells to increase and resemble that observed with liver preparations. Before treatment with salt, the inhibitory activity seems to be physically associated with the binding activity. Adding the isolated inhibitory component back to a suitably activated binding preparation from HTC cells results in a decrease in the apparent affinity for cyclic AMP. The isolated inhibitory component is devoid of cyclic AMP-binding and cyclic AMP phosphodiesterase activities and has an apparent minimal molecular weight of about 30,000 by gel filtration. It possesses protein kinase activity and seems to be identical to the catalytic subunit of a cyclic AMP-stimulated protein kinase on the basis of chromatographic properties and sensitivities to heat and low pH. This catalytic subunit represents only a minor portion of total cellular protein kinase activity and is also present in liver extracts. However, the binding activity from liver is not inhibited significantly under conditions where the binding from HTC cells is affected by the catalytic subunit. The difference in this inhibitory response between liver and HTC preparations appears to reflect differences in the cyclic AMP-binding proteins themselves.     

The separation, properties and possible subunit composition of adenosine 3',5'-monophosphate-dependent protein kinases in brown adipose tissue.

Two 8.5-S protein kinases (ATP : protein phosphotransferase EC 2.7.1.37) and one 6.6-S protein kinase were purified 500--1000-fold from the acid-soluble fraction of brown adipose tissue. The catalytic properties of the kinases were similar. Each kinase was activated by cyclic AMP and had two components of cyclic AMP binding. In the presence of 200 nM cyclic AMP, undissociated kinase activity sedimented at 7.7 or 5.5 S. Free catalytic activity (3.2 S) could be detected but was unstable. Free regulatory units could not be detected. The 8.5-S protein kinase was dissociated by freezing and thawing to a 7.7-S variety with loss of the higher affinity component of binding. The 7.7-S kinase was sedimented through linear gradients of sucrose containing different concentrations of cyclic AMP. At each concentration, kinase activity lost from the holoenzyme peak (% of original) was identical with the amount of cyclic AMP bound at equilibrium (% oof maximum). Similar experiments on the 8.5-S kinase showed that the binding component with higher affinity was not associated with the release of catalytic activity. The results were consistent with the propostal that the kinases isolated contained one more cyclic AMP binding subunit than catalytic subunit (3 : 2 for 8.5 S and 2 : 1 for 6.6 S) and that this extra subunit was released to give an equal number of subunits of each type before catalytic activity was liberated.     

Epinephrine effects on cyclic AMP-dependent protein kinases from rat diaphragms

Diaphragm extracts were subjected to electrophoresis on polyacrylamide gels to separate the different molecular species of th cyclic AMP-dependent protein kinase. Using cyclic [3H]AMP, three peaks of binding activity were observed. The peak closest to the origin (peak I) was associated with cyclic AMP-dependent protein kinase activity and was abolished by incubation of the extracts with cyclic AMP prior to electrophoresis. The peak farthest from the origin (peak III) was devoid of kinase activity and was increased by incubation of extracts with cyclic AMP before electrophoresis; furthermore, when extracts were incubated with cyclic [3H]AMP before electrophoresis, essentially all the radioactivity appeared in peak III. Peak II, in an intermediate position, was also abolished by preincubation of the extracts with cyclic AMP and both its binding capacity and cyclic AMP-dependent protein kinase activity were lower than in Peak I. A peak of cyclic AMP-independent protein kinase (peak 0) that migrated more slowly than peak II was also detected. From these and other data it is concluded that peaks I and II are cyclic AMP-dependent protein kinase and that peak III is the dissociated regulatory subunit, respectively. Peak 0 is cyclic AMP-independent protein kinase together with free catalytic subunits from cyclic AMP-dependent protein kinase. Incubation of rat diaphragms with epinephrine resulted in dose- and time-dependent decrease in peak I and increase in peak III. These changes correlated with the decrease of cyclic AMP-dependent protein kinase associated with peak I. No changes in Peak II were observed with epinephrine, but an increased peak 0 was noted. Changes in peak I and peak III correlated with the modification of glycogen synthase and glycogen phosphorylase activities. No regulatory subunits (peak III) were detected as phosphorylated forms in diaphragms previously equilibrated with 32P. Treatment with epinephrine produce no noticeable phosphorylation of these regulatory subunits.     

Cyclic AMP and mitogen-activated protein kinases are required for glutamate-dependent cyclic AMP response element binding protein and Elk-1 phosphorylation in the dorsal striatum in vivo

Dopaminergic and glutamatergic signalling cascades are integrated in striatal medium spiny neurones by cyclic AMP response-element binding protein and Elk-1 phosphorylation. Phosphorylated cyclic AMP response-element binding protein and phosphorylated Elk-1 contribute to c-fos expression by binding to the calcium and cyclic AMP response-element and the serum response element, respectively, in the c-fos promoter. The role of cyclic AMP and mitogen-activated protein kinase signalling cascades in glutamate-induced cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression was investigated using semiquantitative immunocytochemistry in vivo. Intracerebroventricular infusion of the sodium channel blocker, tetrodotoxin, decreased the glutamate-induced increase in phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Intracerebroventricular infusion of the mitogen-activated and extracellular signal-regulated kinase inhibitor, PD98059, the p38 mitogen-activated protein kinase inhibitor, SB203580, or the cyclic AMP inhibitor, Rp-8-Br-cAMPS, decreased glutamate-induced phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Simultaneous infusion of glutamate and Sp-8-Br-cAMPS, a cyclic AMP analogue, augmented induction of Fos immunoreactivity but not phosphorylated cyclic AMP response-element binding protein or phosphorylated Elk-1 immunoreactivity. These data indicate that cyclic AMP and mitogen-activated protein kinase signalling cascades are necessary for glutamate to induce cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression in the striatum. Furthermore, neuronal activity plays an important role in glutamate-induced signalling cascades in vivo.     

Cyclic AMP-dependent histone-specific nucleoplasmic protein kinase from rat liver.

A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart.     

Physiological Phosphorylation of Protein Kinase A at Thr-197 Is by a Protein Kinase A Kinase

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [³⁵S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low K_m for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [γ-³²P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.     

Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells.

Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.     

Studies on the role of adenosine 3':5'-monophosphate in the activation of liver phosphorylase.

Crude extracts of rabbit liver, preincubated to promote the dephosphorylation of enzymes or other regulatory proteins, were used to study the role of cyclic AMP in the activation of glycogen phosphorylase. Inasmuch as endogenous liver phosphorylase was irreversibly altered by the preincubation procedure, crystalline skeletal muscle phosphorylase was used as the substrate in these studies. In the presence of magnesium ions and ATP, phosphorylase b was converted to phosphorylase a, and in an apparent biphasic process the phosphorylase a formed was subsequently converted to phosphorylase b. In the presence of adenosine 3':5'-monophosphate the rate of phosphorylase a formation and the maximal amount of phosphorylase a formed were increased. The cyclic AMP effect was enhanced by glucose-6-P and required the presence of glycogen. The catalytic subunit of cyclic AMP-dependent protein kinase could replace cyclic AMP in the stimulation of phosphorylase a formation. The effects of cyclic AMP or the catalytic subunit were shown to be due to stimulation of phosphorylase kinase rather than to inhibition of phosphorylase phosphatase. Preliminary fractionation experiments showed that it is possible to separate phosphorylase kinase catalytic activity from a factor or factors required for stimulation of its activation by the catalytic subunit.     

Adenosine cyclic 3',5'-monophosphate-dependent protein kinase from human platelets.

A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit. The apparent Km for ATP in the presence of 10 muM Mg2+ was 4 muM (plus cyclic AMP) and 4.3 muM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 muM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 muM (presence of MgATP) were exhibited by the "protein kinase-cyclic AMP complex". The enzyme required Mg2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate. In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated.