Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Ligation of restriction endonuclease-generated DNA fragments using immobilized T4 DNA ligase

Phosphorylation of terminal deoxynucleotidyl transferase within leukemic cells has been demonstrated, using ³²P labelling of intact cells in culture, followed by immunoprecipitation of the cellular extracts using an anti-terminal transferase antiserum. The phosphate linkage was found to involve serine and threonine residues. Purified calf thymus terminal transferase served as a substrate for cyclic AMP independent protein kinase obtained from leukemic cells. Phosphorylation $\text{in vitro}$ of terminal transferase was accompanied by increased activity and decreased inhibition by excess ribo-ATP. These results indicate that terminal transferase is a physiologic cyclic AMP independent protein kinase substrate, and that this reaction may be important in its control.     

Identification of the regulatory subunit of a cAMP-dependent protein kinase in Dictyostelium discoideum

The occurrence of a cytosolic cAMP-binding protein of an approximate molecular weight of 41,000 daltons was monitored in vegetative and developing amoebae of $\text{Dictyostelium}\text{discoideum}$ by the use of the photoaffinity probe (³²P) 8N₃-cAMP. There was a large apparent increase in the amount of this binding protein during development; its molecular weight remained constant, if appropriate methods were employed for the disruption of the amoebae. Comigration during electrophoresis on two-dimensional gels identifies this cAMP-binding protein, photoaffinity-labeled in crude extracts, as the regulatory subunit of the cAMP-dependent protein kinase of $\text{D.}\text{discoideum}$.     

Cyclic AMP stimulated phosphorylation of liver pyruvate kinase in hepatocytes.

The addition of glucagon (10^?6 M) to an incubation mixture containing ³²Pi and hepatocytes isolated from livers of rats fed $\text{ad libitum}$ results in both a 3-fold increased incorporation of ³²P into L-type pyruvate kinase and a decreased catalytic activity. The ³²P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10^?3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10^?7 M) had essentially no effect on the incorporation of ³²P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase $\text{invivo}$ is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.     

Adenosine 3',5' cyclic monophosphate in Euglena gracilis

$\text{Euglena gracilis}$ contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. $\text{Euglena}$ also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg.     

Some biochemical properties of alkyl phosphotriesters of cyclic AMP.

The biochemical properties of several alkyl phosphotriesters of cyclic AMP were studied with respect to their interactions with beef heart protein kinase and cyclic nucleotide phosphodiesterase. Ethyl and propyl triesters did not enhance the phosphorylation of histone by protein kinase and methyl, ethyl, propyl and butyl triesters were poor competitors for the cyclic AMP binding site of the enzyme. However, these alkyl phosphotriesters were effective inhibitors of cyclic nucleotide phosphodiesterase with the K$\text{i}$'s arrayed in the following order: methyl > ethyl > propyl > butyl > cetyl triester. Metabolic studies with mice indicated that intraperitoneal injection of low doses of propyl triester for one week significantly increased cyclic AMP concentration.     

Inactivation of cyclic GMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethylketone

Cyclic GMP-dependent protein kinase from bovine lung and cyclic AMP-dependent protein kinase from bovine heart are inactivated by N^α-tosyl-L-lysine chloromethylketone (TLCK) in the presence of cyclic GMP and cyclic AMP, respectively. The inactivation of both protein kinases is pseudo-first order, suggesting the rate limiting step is beyond the binding of TLCK. Cyclic GMP-dependent protein kinase is inactivated less than $\text{1}\text{4}$ as rapidly as cyclic AMP-dependent protein kinase, although it shows a higher apparent affinity for TLCK. Cyclic AMP stimulated the rate of inactivation of cyclic AMP-dependent protein kinase 10-fold but cyclic GMP stimulated the rate of inactivation of cyclic GMP-dependent protein kinase only 1.5-fold. The rate of inactivation of cyclic GMP-dependent protein kinase by TLCK is sufficiently rapid (half-time of about 30 min at 37°C with 2 mM TLCK) to account for the effects of TLCK on cell growth observed by others.     

An unusual cyclic AMP-dependent protein kinase from nematodes

The search for an unusual cyclic nucleotide-dependent protein kinase in nematodes represented an attempt to gain some insight into the proposed homology of the cAMP and cGMP-dependent protein kinases. Two species of protein kinase were found in high speed supernatants of the mycophagous nematode $\text{Aphelenchus}$$\text{avenae}$. One of the two, bound to DEAE cellulose and was eluted from it in a manner characteristic of the type I cAMP kinase. The enzyme had high affinity for cAMP and dissociated upon binding to the cyclic nucleotide, as judged by the fact that catalytic activity did not bind to a cAMP affinity column. The second enzyme did not bind to DEAE. Unexpectedly, it too had high affinity for cAMP and much lower affinity for cGMP (unlike the $\text{cAMP}\text{cGMP}$ kinase from insects). The holoenzyme bound tightly to the cAMP affinity column and required a high concentration of the cyclic nucleotide for elution. This latter enzyme is the only example of a cAMP-dependent protein kinase that does not dissociate upon activation.     

The effects of cyclic AMP on disaggregation-induced changes in activities of developmentally regulated enzymes in Dictyostelium discoideum.

The addition of cyclic AMP to the shaking medium of cells disaggregated from pseudoplasmodia of $\text{Dictyostelium discoideum}$ suppressed the accumulation of cell-bound phosphodiesterase which normally occurs (1) after disaggregation. The suppression was not secondarily brought about by its possible inhibitory effect of cyclic AMP on protein synthesis or by its stimulating effect on the release of the enzyme into the medium. The effect was reversible and specific to cyclic AMP. On the other hand, the inhibitory effect of cyclic AMP on the disaggregation-induced inactivation of UDP-galactose transferase was not apparent in the initial period, but thereafter it slowed down the decrease in the enzyme activity. These results indicate that exogenous cyclic AMP mimics at least in part the regulatory effects of cell-to-cell contact on certain enzymes.     

Cyclic AMP-dependent phosphorylation of rat liver 6-phosphofructo 2-kinase, fructose 2,6-bisphosphatase

Incorporation of ³²P from [γ-³²P]ATP into a homogeneous preparation of rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was catalyzed by a homogeneous preparation of the catalytic subunit of the cyclic AMP dependent protein kinase from rat liver. Approximately 2 mol of phosphate were incorporated per mol of the dimeric enzyme and this was associated with inhibition of the phosphotransferase activity and activation of the phosphohydrolase activity. Acid hydrolysis of the enzyme that was phosphorylated $\text{in}$,$\text{vitro}$ revealed that only seryl residues were labeled. Fructose 2,6-bisphosphate inhibited the initial rate of phosphorylation of the enzyme. It is concluded that both activities of this bifunctional enzyme are regulated in a reciprocal manner by cyclic AMP-dependent phosphorylation and that this phosphorylation can be modulated by fructose 2,6-bisphosphate.     

Regulation of guanylate cyclase activity during cytodifferentiation of Blastocladiella emersonii.

Levels of guanylate cyclase activity in extracts of the unicellular eukaryote $\text{Blastocladiella}$$\text{emersonii}$ differed by at least 100-fold at different stages of the cell cycle, paralleling changes in the cyclic GMP content of this organism (Proc. Natl. Acad. Sci. U.S.A. $\text{72}$, 442 (1975)). Extracts of vegetative cells lacked appreciable guanylate cyclase activity, whereas the specific activity of the enzyme in zoospore extracts was 2 nmol cyclic GMP synthesized/min/mg protein at 35°. Guanylate cyclase activity increased at least 50-fold during the period of zoospore formation when cyclic GMP begins to accumulate $\text{in}$$\text{vivo}$. Since actinomycin D or cycloheximide added at the beginning of this period blocked any increase in enzyme activity, it appears that $\text{de}$$\text{novo}$ synthesis of guanylate cyclase during sporulation is responsible for the accumulation of cyclic GMP that occurs at that time.     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.     

Protein kinase activity in mouse mammary carcinoma.

C3H mouse mammary carcinoma contains cyclic AMP-independent (C) and dependent (RC) protein kinases and a specific cyclic AMP-binding protein (R). The specific activities of C, RC and R are markedly lower in carcinoma than the normal mammary cells. Protein kinase preparation from neoplastic cells showed markedly higher ration of $\text{C}\text{RC}$ and lower responsiveness to cyclic AMP for the activation of the enzyme than the normal cells.     

An effect of diet on the activity of phosphofructokinase in rat heart

Phosphorylation of the inhibitory subunit of cardiac troponin (TN-I) occurs $\text{in vivo}$ after catecholamine intervention through adenylcyclase, cyclic AMP and cyclic AMP dependent protein kinase system. Also, TN-I and tropomyosin binding subunit of troponin (TN-T) are specifically hydrolyzed by calcium-activated neutral protease (CANP). In this study, we compared proteolysis of a set of TNs before and after phosphorylation by cyclic AMP dependent protein kinase plus cyclic AMP, using CANP from cardiac muscle. The initial rate of peptide release from both TNs was the same. After prolonged incubation, however, unphosphorylated TN degradation retarded, while phosphorylated TN proteolysis still continued. The amount of peptide release at the latter phase was dependent on the degree of phosphorylation. These results were confirmed by SDS polyacrylamide gel electrophoresis, and they suggest that a conformational change occurred in the whole TN molecule after phosphorylation of TN-I.     

In vivo activation of cyclic adenosine 3':5'-phosphate-dependent protein kinase in Chinese hamster ovary cells treated with N-6, O-2'-diburyl cyclic adenosine 3':5'-phosphate.

Treatment of Chinese hamster ovary cells with dibutyryl cyclic AMP, which results in a net increase of the intracellular cyclic AMP level, converts the epithelial-like cells to a fibroblast-like shape. Protein kinase activity in cells treated with 1 mM dibutyryl cyclic AMP show a 3-fold increase in V_max but no appreciable changes in the apparent K_m for ATP. When cells are treated with dibutyryl cyclic AMP, there is a time-dependent conversion of cyclic AMP-stimulable protein kinase to cyclic AMP-independent catalytic subunits, as demonstrated by Sephadex G-100 gel filtration. These experiments demonstrate the activation of the cyclic AMP-dependent protein kinase $\text{in vivo}$. This activation may lead to phosphorylation of certain cellular constituent(s) and thus may be involved in the observed morphological transformation.     

Cyclic AMP-dependent inactivation of human liver pyruvate kinase.

Human liver pyruvate kinase is rapidly (within 2 min) inactivated by incubation of a human liver supernatant with cyclic AMP, when measured at suboptimal substrate concentrations. Half-maximal inactivation is reached with 0.04 μM cyclic AMP. The apparent K_0.5 for phosphoenolpyruvate shifts from 0.5 mM to 1.1 mM by incubation with cyclic AMP. It is concluded that cyclic AMP-dependent protein kinase may catalyze the phosphorylation of human liver pyruvate kinase $\text{in vivo}$.     

Regulation of rat brain (Na+ + K+)-ATPase activity by cyclic AMP

The interaction between the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5′-AMP, cyclic GMP or 5′-GMP, could inhibit the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854–3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, resulted in a decrease in overall $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has no effect on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme, there appeared to be a decrease in the Na⁺-dependent phosphorylation of the Na⁺-ATPase enzyme, while the K⁺-dependent dephosphorylation of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was unaffected.     

Activation of protein kinase in the guinea pig fundic gastric mucosa by histamine.

The effect of histamine, 1,4-methylhistamine and ethanol on cyclic AMP levels and protein kinase activation was measured in tissue strips from the fundic region of guinea pig gastric mucosa. Histamine induced a significant elevation of tissue cyclic AMP levels and also $\text{in situ}$ activation of the protein kinase. 1,4-methylhistamine, an inactive analog of histamine, and ethanol had no effect on these two parameters. Results suggest that protein kinase activation is involved in the cyclic AMP-mediated action of histamine on the gastric fundic mucosa.     

Adenosine 3′, 5′-cyclic monophosphate in Schistosoma mansoni

Homogenates of adult $\text{Schistosoma mansoni}$ (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E₁, E₂ or B₁. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1×10}{}^{\mathrm{?7}}\text{M}$) over cyclic GMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5×10}{}^{\mathrm{?6}}\text{M}$). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen.     

In vivo regulation of hepatic protein kinase by adenosine 3',5'-monophosphate mediated glucagon stimulation

$\text{In vivo}$ administration of glucagon caused an increase in the dissociation of protein kinase subunits which was accompanied by elevated adenosine 3′,5′-monophosphate concentrations in the rat liver. Concomitantly, there was a decrease in non saturated adenosine 3′,5′-monophosphate binding sites. A reduction in protein kinase activity measured in the presence of the cyclic nucleotide was apparent at 5 minutes of glucagon administration while enzyme activity assayed in the absence of adenosine 3′,5′-monophosphate was already increased after one minute. Glucose, given through an intragastric tube, caused no changes in the effect of glucagon on hepatic protein kinase.