Inhibitory effect of a lethal toxic fragment of staphylococcal alpha-toxin on cyclic AMP-dependent protein kinase activity.

The effect of a lethal toxic fragment of staphylococcal alpha-toxin on the activity of adenosine 3',5'-monophosphate(cyclic AMP)-dependent protein kinase was examined. 1. The lethal toxic fragment produced a dose-dependent decrease in both the binding of cyclic AMP to the regulatory subunit and phosphorylation activity of cyclic AMP-dependent protein kinase obtained from rabbit skeletal muscles up to a plateau at a 50% inhibitory effect. The decrease in the activity of protein kinase observed with low doses of the lethal toxic fragment (0.1 microM) resulted from a competitive inhibition, probably by its interaction with the cyclic AMP-binding site in the regulatory subunit molecule. 2. The effects of a lethal toxic fragment and epinephrine on the cyclic AMP level and protein kinase activity were investigated in the perfused rabbit heart slices. The lethal toxic fragment attenuated the stimulation of cyclic AMP-dependent protein kinase activity ratio by epinephrine. 3. It is suggested that the specific action of a lethal toxic fragment on the cellular membrane enzymes may be attributable to the inhibition of the cyclic AMP-dependent protein kinase activity.     

Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia

Studies were carried out to elucidate the mechanisms underlying the diminished phosphorylation of cerebral ribosomal protein in experimental hyperphenylalaninaemia [Roberts & Morelos (1980) Biochem. J.190, 405-419]. Administration of N(6),O(2)'-dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine, which increased phosphorylation of the S6 protein of cerebral 40S ribosomal subunits in control infant rats, did not counteract the decreased phosphorylation of this ribosomal protein resulting from intraperitoneal administration of a loading dose of l-phenylalanine. N(2),O(2)'-Dibutyryl cyclic GMP had no effect on cerebral ribosomal-protein phosphorylation in either control or hyperphenylalaninaemic animals. The phenylalanine-induced decrease in ribosomal-protein phosphorylation was associated with decreased protein kinase activity in cerebral cytosolic and microsomal preparations. However, the maximal protein kinase response to cyclic AMP added in vitro was unaltered by prior administration of phenylalanine in vivo. The heat-stable protein inhibitor of cyclic AMP-dependent protein kinases decreased the activity of these enzymes by about 90% and eliminated the phenylalanine-induced difference in protein kinase activity in the absence of added cyclic AMP. Intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine which increased the cyclic AMP-dependent protein kinase activity ratio in control infant rats was without effect on this index in phenylalanine-treated animals. Dibutyryl cyclic GMP had no effect on the protein kinase activity ratio in either group of animals. These results suggest that inhibition of cerebral cyclic AMP-dependent protein kinases by abnormally high concentrations of phenylalanine may contribute to the decrease in cerebral ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.     

The release of superoxide anion from granulocytes: effect of inhibitors of anion permeability.

In vivo administration of ecdysterone produced a decrease in cyclic AMP levels and cyclic AMP-binding protein activity in mouse liver 40 min after injection. These changes were accompanied by a concomitant decrease in cyclic AMP-dependent protein kinase. The effect on phosphoprotein phosphatase was the opposite pattern of that on protein kinase. These results support the idea that the cyclic AMP-protein kinase system may be involved in the heterophylic action of ecdysterone.     

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates. Relationship with phosphorylation

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20-30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.     

Effects of adenosine 3':5'-monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac and slow and fast contracting skeletal muscles.

Effects of cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase were studied in sarcoplasmic reticulum prepared from cardiac and slow and fast (white) skeletal muscle. Cyclic AMP-dependent protein kinase failed to catalyze phosphorylation of fast skeletal muscle microsomes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyclic AMP-dependent protein kinase was without effect on calcium uptake by these microsomes. Treatment of cardiac microsomes obtained from dog, cat, rabbit, and guinea pig with cyclic AMP-dependent protein kinase and ATP resulted in phosphorylation of a 22,000-dalton protein component in the amounts of 0.75, 0.25, 0.30, and 0.14 nmol of phosphorus/mg of microsomal protein, respectively. Calcium uptake by cardiac microsomes was stimulated 1.8- to 2.5-fold when microsomes were treated with cyclic AMP-dependent protein kinase. Protein kinases partially purified from bovine heart and rabbit skeletal muscle were both effective in mediating these effects on phosphorylation and calcium transport in dog cardiac sarcoplasmic reticulum. Slow skeletal muscle sarcoplasmic reticulum also contains a protein with a molecular weight of approximately 22,000 that can be phosphorylated by protein kinase. Phosphorylation of this component ranged from 0.005 to 0.016 nmol of phosphorous/mg of microsomal protein in dog biceps femoris. A statistically significant increase in calcium uptake by these membranes was produced by the protein kinase. Increases in protein kinase-catalyzed phosphorylation of a low molecular weight microsomal component and in calcium transport by sarcoplasmic reticulum of cardiac and slow skeletal muscle may be related to the relaxation-promoting effects of epinephrine seen in these types of muscle. Conversely, the absence of a relaxation-promoting effect of epinephrine in fast skeletal muscle may be associated with the lack of effect of cyclic AMP and protein kinase on calcium transport by the sarcoplasmic reticulum of this type of muscle.     

Induction of ornithine decarboxylase by biliverdin in rat liver.

The administration of biliverdin (0.1mg/g of body weight) into the peritoneal cavity of rats resulted in the induction of ornithine decarboxylase in the liver. When the temporal relationships between the changes in intracellular adenosine 3', 5'-cyclic monophosphate (cyclic AMP) level, cyclic AMP-dependent protein kinase activity and the induction of ornithine decarboxylase were investigated, the concentration of cyclic AMP increased significantly 2 h after the administration of biliverdin, while cyclic AMP-dependent protein kinase was activated after 2–4 h. The hepatic ornithine decarboxylase activity began to increase 4 h after biliverdin injection. These results suggest that there is some sequential relationship between the increase of cyclic AMP, the activation of cyclic AMP-dependent protein kinase and the induction of ornithine decarboxylase although the direct correlation of these three events remains to be elucidated.     

Purification and general properties of guanosine 3':5'-monophosphate-dependent protein kinase from guinea pig fetal lung.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was purified from the guinea pig fetal lung, a tissue shown to be the richest in this enzyme in all mammalian sources examined, and its general properties studied. The enzyme was purified 150-fold from crude extract by steps of pH 5.4 isoelectric precipitation, Sephadex G-200 filtration, hydroxylapatite treatment and DEAE-cellulose chromatography. The purified enzyme, free from contamination with adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase, had a specific activity at least equivalent to 600-fold purification of the enzyme from the adult lung. The pulmonary enzyme exhibited an absolute requirement of protein kinase modulator (prepared from various mammalian tissues with an exception of skeletal muscle) for its activity. Inhibitor protein of cyclic AMP-dependent protein kinase purified from rabbit skeletal muscle could not stimulate nor inhibit the cyclic GMP target enzyme, indicating the factors from mammalian sources regulating the two classes of protein kinases may not be the same. The enzyme had Ka values of 1.3 times 10(-8) and 3.3 times 10(-8) M for 8-bromo cyclic GMP and cyclic GMP, respectively, compared to 3.0 times 10(-6) M for cyclic AMP. Cyclic GMP lowered the Km of the enzyme for ATP from 6.3 times 10(-5) M in its absence to 2.1 times 10(-5) M in its presence, accompanied by an approximate doubling of the Vmax. The molecular weight of the enzyme (assayed by its catalytic and cyclic GMP-binding abilities) was estimated to be 123,000, corresponding to a sedimendation coefficient of 7.06 S, by means of sucrose density gradient ultracentrifugation. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity with optimal concentrations of about 30 and 0.7 mM, respectively. The maximal activity seen in the presence of Mg2+, however, was nearly twice as high as that seen in the presence of Co2+. Histones were generally effective substrates for the enzyme, whereas protamine, casein, phosvitin, phosphorylase kinase, and activator protein of phosphodiesterase were not. The cyclic GMP-dependent enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent enzyme in the presence of Mg2+.     

Effect of cyclic AMP-dependent protein kinase on insulin receptor tyrosine kinase activity.

To explain the insulin resistance induced by catecholamines, we studied the tyrosine kinase activity of insulin receptors in a state characterized by elevated noradrenaline concentrations in vivo, i.e. cold-acclimation. Insulin receptors were partially purified from brown adipose tissue of 3-week- or 48 h-cold-acclimated mice. Insulin-stimulated receptor autophosphorylation and tyrosine kinase activity of insulin receptors prepared from cold-acclimated mice were decreased. Since the effect of noradrenaline is mediated by cyclic AMP and cyclic AMP-dependent protein kinase, we tested the effect of the purified catalytic subunit of this enzyme on insulin receptors purified by wheat-germ agglutinin chromatography. The catalytic subunit had no effect on basal phosphorylation, but completely inhibited the insulin-stimulated receptor phosphorylation. Similarly, receptor kinase activity towards exogenous substrates such as histone or a tyrosine-containing copolymer was abolished. This inhibitory effect was observed with receptors prepared from brown adipose tissue, isolated hepatocytes and skeletal muscle. The same results were obtained on epidermal-growth-factor receptors. Further, the catalytic subunit exerted a comparable effect on the phosphorylation of highly purified insulin receptors. To explain this inhibition, we were able to rule out the following phenomena: a change in insulin binding, a change in the Km of the enzyme for ATP, activation of a phosphatase activity present in the insulin-receptor preparation, depletion of ATP, and phosphorylation of a serine residue of the receptor. These results suggest that the alteration in the insulin-receptor tyrosine kinase activity induced by cyclic AMP-dependent protein kinase could contribute to the insulin resistance produced by catecholamines.     

Glycogen synthase activity in Chinese hamster ovary cells: Studies with wild-type and mutant cells defective in cyclic AMP-dependent protein kinase

Glycogen synthase (EC 2.4.1.11) activity was studied in cell extracts from wild-type Chinese hamster ovary (CHO) cells and three mutants resistant to cyclic AMP effects on cell shape and cell growth. Based on the capacity of crude extracts to phosphorylate exogenous hisone, two of the mutants appeared to have altered cyclic AMP-dependent protein kinase (EC 2.7.1.37) and one of them had apparently normal amounts of kinase activity. Glycogen synthase activity was present in comparable amounts in wild-type and all three mutant strains in a presumably inactive phosphorylated form since activity was virtually completely dependent upon the presence of glucose 6-phosphate. The enzyme could be partially dephosphorylated by endogenous phosphatases and rephosphorylated by exogenous cyclic AMP-dependent protein kinase. Attempts to find culture conditions (e.g. glucose starvation)_or cell treatment (e.g. insulin) which might activate glycogen synthase in intact cells were unsuccessful. Since glycogen synthase activity present in CHO cells was independent of the level of cyclic AMP-dependent kinase, we conclude that cyclic AMP-dependent protein kinase does not play a critical role in regulating the state of phosphorylation of the synthase.     

Glycogen synthase activity in Chinese hamster ovary cells. Studies with wild-type and mutant cells defective in cyclic AMP-dependent protein kinase

Glycogen synthase (EC 2.4.1.11) activity was studied in cell extracts from wild-type Chinese hamster ovary (CHO) cells and three mutants resistant to cyclic AMP effects on cell shape and cell growth. Based on the capacity of crude extracts to phosphorylate exogenous histone, two of the mutants appeared to have altered cyclic AMP-dependent protein kinase (EC 2.7.1.37) and one of them had apparently normal amounts of kinase activity. Glycogen synthase activity was present in comparable amounts in wild-type and all three mutant strains in a presumably inactive phosphorylated form since activity was virtually completely dependent upon the presence of glucose 6-phosphate. The enzyme could be partially dephosphorylated by endogenous phosphatases and rephosphorylated by exogenous cyclic AMP-dependent protein kinase. Attempts to find culture conditions (e.g. glucose starvation) or cell treatment (e.g. insulin) which might activate glycogen synthase in intact cells were unsuccessful. since glycogen synthase activity present in CHO cells was independent of the level of cyclic AMP-dependent kinase, we conclude that cyclic AMP-dependent protein kinase does not play a critical role in regulating the state of phosphorylation of the synthase.     

The use of affinity chromatography in purification of cyclic nucleotide receptor proteins.

Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.     

Hormonal regulation of glycogen synthase: Insulin decreases protein kinase sensitivity to cyclic AMP

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1–2 μM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 μM. Insulin (0–100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.     

Comparison of cyclic nucleotide specificity of guanosine 3′,5′-monophosphate-dependent protein kinase and adenosine 3′,5′-monophosphate-dependent protein kinase

Guanosine 3′,5′-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3′,5′-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-folds less than that of cylic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic. AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophophorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Lymphocyte protein kinase activity in cells from young and elderly men and women

Protein kinase activity of lymphocytes isolated from human subjects was assayed using histone as substrate. The activity was stimulated about twofold by cyclic AMP and total enzyme activity, determined in the presence of cyclic AMP, was inhibited by 65% by the specific heat-stable inhibitor of cyclic AMP-dependent protein kinase. Histone phosphorylation was not stimulated by cyclic GMP in the presence of the inhibitor. Cyclic AMP-dependent protein kinase could be activated in vitro by incubating intact cells with isoproterenol or with forskolin and was reflected by a significant (P less than 0.05) increase in the protein kinase activity ratio. In contrast to these well-characterized adenylate cyclase activators, incubating cells for up to 2 hr in vitro in the presence of the specific beta-blocker propranolol had no significant effect on the amount of cyclic AMP-dependent protein kinase that was in the activated state. When compared in subjects between the ages of 21 and 74 years, lymphocyte protein kinase activity was unaltered by age or gender. These results indicate that cyclic nucleotide-dependent protein kinase is of the cyclic AMP-dependent variety in the human lymphocyte. A low amount of the cyclic AMP-dependent activity (about 15%) is in the already activated state in freshly isolated cells, and this is not further reduced by incubation in vitro or by beta-blockade. In contrast to previously reported changes in the capacity to synthesize cyclic AMP, lymphocyte protein kinase is unaltered by gender or age in human subjects.     

Two types of cyclic GMP binding site associated with the cyclic AMP-dependent protein kinase from lymphocytes.

Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site. The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP. Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [3H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (Kd about 4 . 10(-8) M) belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (Kd 2--5 . 10(-6) M) was demonstrated by the inhibitory effect of 10(-5) M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site.     

Glycogen synthase kinases. Distribution in mammalian tissues of forms that are independent of cyclic AMP.

Extracts of rat tissues contain kinases which catalyze the conversion of glycogen synthease from the glucose 6-phosphate-independent (I) form to the glucose 6-phosphatate-dependent (D) form. These kinases were stimulated by adenosine 3':5' monophosphate (cyclic AMP). The glycogen synthase kinase activity ratio (activity in the absence of cyclic AMP divided by activity in the presence of cyclic AMP) varied from 0.28 to 0.97. The activity ratio for histone kinase in the same extracts ranged from 0.11 to 0.29. The levels of glycogen synthase kinase varied by a factor of 80 in the following rat tissues (given in order of decreasing enzyme activity): kidney, liver, stomach mucosa, lung, brain, heart, skeletal muscle, and adipose tissue. In the same tissues the levels of histone kinase varied by only a factor of 6 and did not correlate with the levels of glycogen synthase kinase. A modification of the method of Walsh et al. ((1971) J. Biol. Chem. 246, 1977-1985) was developed for purification of the heat-stable inhibitor of cyclic AMP-dependent protein kinases (inhibitor). The modified procedure resulted in good yields of highly purified inhibitor and was much simpler than the previously described procedure. This inhibitor completely inhibited cyclic AMP-dependent histone kinase activity of the extracts but much of the glycogen synthase kinase activity was not inhibited. The portion of glycogen synthase kinase that was insensitive to the inhibitor was: stomach mucosa, 95%; brain, 90%; liver, 82%; kidney, 81%; lung, 68%; adipose tissue, 65%; skeletal muscle, 63%; and heart, 54%. This histone kinase activity in the extracts and hte ratio of glycogen synthase kinase to histone kinase activity of purified catalytic subunit of the cyclic AMP-dependent protein kinase was used to calculate for each extract the glycogen synthase kinase activity contributed by the cyclic AMP-dependent protein kinase. Based on these calculations, the portion of the glycogen synthase kinase which was due to kinases independent of cyclic AMP was: kidney, 97%; liver, 91%; lung, 89%; brain, 87%, heart, 85%; stomach mucosa, 84%; adipose tissue, 38%; and skeletal muscle, 33%. A significant portion of the glycogen synthase kinase activity, but virtually none of the cyclic AMP-dependent histone kinase activity, of these extracts could be adsorbed to phosphocellulose columns. Liver extracts contained, in addition, a form of glycogen synthase kinase which was not adsorbed to phosphocellulose and which could be separated from the cyclic AMP-dependent protein kinase by additional chromatography. These studies demonstrate that kinases independent of cyclic AMP account for most of the glycogen synthase kinase activity of many tissues. The widespread distribution and high concentrations of these enzymes suggest that they are of physiological importance.     

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.     

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.     

Developmental changes in cyclic AMP, protein kinase, phosphorylase kinase, and phosphorylase in liver, heart, and skeletal muscle of the rat

Changes in cyclic AMP, protein kinase, phosphorylase kinase, and phosphorylase levels were examined during development in the rat. In liver, cyclic AMP increased prenatally and for the first 10 postnatal days; protein kinase levels (both cyclic AMP-dependent and independent activities) were high prenatally and declined during the first 10 postnatal days. Both phosphorylase and phosphorylase kinase in liver increased rapidly prenatally and more slowly postnatally. In heart and skeletal muscle cyclic AMP increased prenatally and for the first 10 days after birth, then declined. Protein kinase in both these tissues was highest prenatally and declined perinatally. In heart and skeletal muscle phosphorylase and phosphorylase kinase activities were extremely low prenatally although both enzymes were largely in their activated forms. Postnatally the nonactive form of both enzymes increased greatly throughout 30 postnatal days. In all three tissues, particularly heart and skeletal muscle, these changes could not be correlated with levels of tissue glycogen.