Activation of cyclic AMP-dependent protein kinase by epinephrine in proliferating lymphoid cells

Activation of the cyclic AMP-dependent protein kinase in intact lymphosarcoma cells can be promoted by epinephrine. The lymphosarcoma protein kinase is approximately 90% Isozyme I. Using the synthetic peptide PK-1 (LeuArgArgAlaSerLeuGly) as substrate for the kinase, the cyclic AMP-dependent protein kinase activity was 95% of the total protein phosphotransferase activity in the cell extract. In control cells the optimum extraction buffer for preventing enzyme subunit dissociation or reassociation contained buffer (2(N-morpholino)ethanesulfonic acid), EDTA, 2-mercaptoethanol, and charcoal. The absence of charcoal or the presence of 0.14 m KCl in the buffer promoted enzyme dissociation in the extract. The phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine had no effect. In extracts from epinephrine-treated cells or extracts to which purified catalytic subunit of the cyclic AMP-dependent protein kinase was added, recovery of the total protein kinase activity was 25% of that predicted in experiments with control cells. Recovery of enzyme activity increased to 80–95% of the predicted value when 0.14 m KCl was included in the extraction buffer. Methods involving a two-buffer extraction procedure are presented as the optimum protocol for determining in vivo activation of the cyclic AMP-dependent protein kinase, Isozyme I. Using these methods, epinephrine (1 μm) dissociated the cyclic AMP-dependent protein kinase essentially 100% in intact lymphosarcoma cells. The dissociation was apparently maintained for up to 60 min. Approximately 10–15% of the dissociated enzyme may be specifically associated with particulate cell fractions. Collectively the data emphasize the experimental difficulty inherent in determination of the extent of in vivo dissociation of the cyclic AMP-dependent protein kinase.     

A cyclic AMP dependent protein kinase in Dictyostelium discoideum

A cyclic AMP-dependent protein kinase was found to appear during the time course of development of $\text{Dictyostelium}\text{discoideum}$. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from $\text{Dictyostelium}$ showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography.     

Action of cyclic nucleotide analgoues in Chinese hamster ovary cells

Cyclic nucleotide analogues have been tested for their ability to cause the morphological conversion of Chinese hamster ovary cells in culture, as well as for effects on cyclic AMP-related enzymes. The ability of the analogues to inhibit the cyclic AMP phosphodiesterase activity and to activate the cyclic AMP-dependent protein kinase activity in cell extracts has been measured. Cell cultures were incubated with the analogues and the effects on morphology, intracellular level of cyclic AMP, and in vivo protein kinase activation were determined. All analogues which induced the morphological conversion also caused in vivo activation of the cyclic AMP-dependent protein kinase. Only N⁶,O^2′-dibutryl and N⁶-monobutyryl cyclic AMp caused caused on increase in intracellular cyclic AMP, presumably through inhibition of the intracellular cyclic AMP phosphodiesterase activity. The increase in cyclic AMP appears to cause the protein kinase activation. However, analogues such as 8-bromo and 8-benzylthio cyclic AMP do not cause any change in intracellular cyclic AMP level and appear to activate the intracellular cyclic AMP-dependent protein kinase directly.     

The effect of growth state on the activity of the protein kinase isoenzymes an increase in type II cyclic AMP-dependent protein kinase is correlated with growth arrest in G1 phase

Native polyacrylamide gels have been used to resolve protein kinase isoenzymes from cultured cells and the protein kinases have been identified by carrying out phosphorylation reactions in the gel. Following electrophoresis, the gels were incubated with histome and [γ-³²P]ATP. The gels were then thoroughly washed and dried down, and the protein kinases were located by autoradiography. Protein kinase activity as measured in the gel system was a linear function of cytosol protein concentration up to about 100 μg per channel and incorporation of ³²P into histone was time dependent. Three bands of protein kinase activity were resolved in cytosol samples from baby hamster kidney (BHK) fibroblasts. The band with the lowest relative mobility utilized histone IIA or casein equally well as substrate protein whereas bands 2 and 3 demonstrated a clear preference for histone. Bands 2 and 3 displayed a relative mobility in electrophoresis that was identical to that observed for cyclic AMP-dependent protein kinases I and II from rat liver. Treatment of cyctosol samples with cyclic AMP prior to electrophoresis resulted in the disappearance of cyclic AMP-dependent protein kinases from the gel profile. This method was employed to identify bands 2 and 3 as cyclic AMP-dependent protein kinases. The protein kinases in growth-arrested cells were compared with proliferating cells. We have observed a 3.5-fold increase in the activity of Type II protein kinase as the cells arrest growth in G₁ phase of the cell cycle. This increase in Type II is correlated with the increase in cells blocked in G₁ and a decrease in II Type activity appears to be an early event in permitting cells to leave G₁ and resume growth.     

Suppression of cyclic AMP-dependent protein kinase activity in murine melanoma cells by 12-0-tetradecanoylphorbol-13-acetate

The effect of the potent tumor promoter 12-0-tetradeconylphorbol-13-acetate (TPA) on the cyclic AMP metabolism of B16 mouse melanoma cells was examined. TPA (10^?7M) slightly increased the growth rate and inhibited melanin production by these cells. Although TPA had little effect on basal or hormone stimulated cyclic AMP levels, it did significantly suppress cyclic AMP-dependent protein kinase activity from treated cells in a dose-dependent fashion. Other phorbol ester and non-phorbol ester tumor promoters also suppressed cyclic AMP-dependent protein kinase activity while the non-promoter, phorbol, did not alter cyclic AMP-dependent protein kinase activity.     

Phosphorylation and activation of acetyl-coenzyme A carboxylase kinase by the catalytic subunit of cyclic AMP-dependent protein kinase

The catalytic subunit of cyclic AMP-dependent protein kinase stimulates the inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase. The stimulated inactivation of carboxylase is due to activation of carboxylase kinase by the catalytic subunit. Activation of carboxylase kinase activity is accompanied by the incorporation of 0.6 mol of phosphate per mole of carboxylase kinase. Addition of the regulatory subunit of cyclic AMP-dependent protein kinase prevents the activation of carboxylase kinase. Phosphorylation and activation of carboxylase kinase has no effect on the K_m for ATP, but decreases the K_m for acetyl-CoA carboxylase from 93 to 45 nm. Inactivation of carboxylase by the carboxylase kinase requires the presence of coenzyme A even when the activated carboxylase kinase is used. Acetyl-CoA carboxylase is not phosphorylated or inactivated by the catalytic subunit of cyclic AMP-dependent protein kinase.     

Altered cyclic AMP-dependent protein kinase activity in a mutant adrenocortical tumor cell line

A somatic cell genetic approach has been used to evaluate the role of cyclic AMP-dependent protein kinase in ACTH action on adrenal steroidogenesis. A mutant clone, 8BrcAMP^r-1, previously was isolated from an ACTH-sensitive adrenocortical tumor cell line (clone Y1) following mutagenesis and selective growth in 8-bromoadenosine 3′, 5′-monophosphate. This study demonstrates that the 8BrcAMP⁴-1 cells have an altered cyclic AMP-dependent protein kinase. The protein kinase in the cytosol of the mutant characteristically requires, for half-maximal activity, concentrations of cyclic AMP 7-fold higher than those required by the enzyme in preparations from the parent. The cytosolic cyclic AMP-dependent protein kinases of Y1 and 8BrcAMP^r-1 cells chromatograph similarly on columns of DEAE-cellulose. From each cell line, a major peak of activity (≥ 70% of recovered activity), designated as Peak I, elutes with 0.04–0.06 M NaCl; a second peak of activity, designated as Peak II, elutes with 0.12–0.14 M NaCl. Protein kinase activity in the Peak I fraction of mutant cells has a decreased apparent affinity (4-fold) for cyclic AMP relative to the corresponding fraction of parental Y1 cells. The protein kinase activities present in Peak II fractions from Y1 and mutant cells are indistinguishable. The protein kinase mutant exhibits poor steroidogenic responses to added ACTH and cyclic AMP; and as shown previously does not display the growth arrest and morphological changes produced in Y1 by these agents. These results suggest that cyclic AMP-dependent protein kinase is important in the regulation of adrenal steroidogenesis, morphology and growth by ACTH.     

Isolation and characterization of a variant of B16-mouse melanoma resistant to MSH growth inhibition

A variant of B-16 F₁ mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition by MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F₁ cells. MSH induced a twofold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F₁ cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F₁ kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F₁ and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F₁ cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.     

Cyclic AMP stimulation of membrane phosphorylation and Ca2+-activated,Mg2+-dependent ATPase in cardiac sarcoplasmic reticulum

Ca²⁺-activated ATPase (EC 3.6.1.15) in canine cardiac sarcoplasmic reticulum was stimulated 50–80% by cyclic adenosine 3′ : 5′-monophosphate. The relationship of this stimulation to cyclic AMP-dependent membrane phosphorylation with phosphoester bands was studied. Cyclic AMP stimulation of ATPase activity was specific for Ca²⁺-activated ATPase and was half-maximal at about 0.1 μM which is similar to the concentration required for half-maximal stimulation of membrane phosphorylation by endogenous cyclic AMP-stimulated protein kinase (EC 2.7.1.37). Cyclic AMP stimulation of Ca²⁺-activated ATPase was calcium dependent and maximal at calculated Ca²⁺ concentrations of 2.0 μM. Cyclic AMP-dependent Ca²⁺-activated ATPase correlated well with the cyclic AMP-dependent membrane phosphorylation of which 80% was 20 000 molecular weight protein identified by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. In trypsin-treated microsomes, cyclic AMP did not stimulate Ca²⁺-activated ATPase or phosphorylation of the 20 000 molecular weight membrane protein. An endogenous calcium-stimulated protein kinase (probably phosphorylase b kinase) with an apparent K_m for ATP of 0.21–0.32 mM was present and appeared to be involved in the cyclic AMP-dependent phosphorylation of the 20 000 molecular weight protein which was calcium dependent. Cyclic guanosine 3′ : 5′-monophosphate did not inhibit any of the stimulatory effects of cyclic AMP. These data suggest that the cyclic AMP stimulation of Ca²⁺-activated ATPase in cardiac sarcoplasmic reticulum is mediated by the 20 000 molecular weight phosphoprotein product of a series of kinase reactions similar to those activating phosphorylase b.     

Protein Kinase Activities in Neurospora crassa

Several protein kinase activities have been found in 105,000g supernatant of Neurospora crassa mycelia grown up to the logarithmic phase. By chromatography on DEAE-cellulose the following enzyme activities have been resolved: (i) a cyclic AMP-dependent protein kinase (peak I kinase) eluting at 0.20 m NaCl, more active with histone than with phosvitin (it was inhibited by both a thermolabile fraction having cyclic AMP-binding activity and a thermostable inhibitor isolated from 105,0005g mycelial supernates), (ii) a cyclic nucleotide-independent protein kinase (peak II kinase) eluting at 0.35 m NaCl, also more active with histone than with phosvitin (this kinase was not inhibited by the fraction having cyclic AMP-binding activity but it was sensitive to the thermostable inhibitor); and finally, (iii) a protein kinase eluting at 0.43 m NaCl (peak II kinase), with similar activity toward histone and phosvitin, insensitive to cyclic nucleotides and to fractions carrying cyclic AMP-binding capacity (this kinase activity also resulted insensitive to the thermostable inhibiting factor).     

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.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743–745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L₆ myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system.We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f₂b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10^?5 M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase.Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems.The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane missing data     

Activation of cyclic AMP-dependent protein kinase and stimulation of protein phosphorylation in response to adenosine in C-1300 murine neuroblastoma

DEAE-cellulose chromatography of the 20,000g supernatant fraction of homogenates of C-1300 murine neuroblastoma (clone N2a) yields one major and two minor peaks of cyclic AMP-dependent protein kinase activity. Assessment of the endogenous activation state of the enzyme(s) reveals that the enzyme is fully activated by the treatment of whole cells with adenosine (10 μM) in the presence of the phosphodiesterase inhibitor Ro 20 1724 (0.7 mM). This treatment produces a large elevation in the cyclic AMP content of the cells. The treatment of whole cells with adenosine alone (1–100 μM) or Ro 20 1724 alone (0.1–0.7 mM) produces minimal elevations in cyclic AMP but nevertheless causes significant activations of cyclic AMP-dependent protein kinase. The autophosphorylation of whole homogenates of treated and untreated cells was studied using [γ-³²P] ATP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Treatments which activate cyclic AMP-dependent protein kinase selectively stimulate the incorporation of ³²P into several proteins. This stimulation is most prominent in the 15,000-dalton protein band. The addition of cyclic AMP to phosphorylation reactions containing homogenate of untreated cells stimulates the phosphorylation of the same protein bands. These results indicate that adenosine may have regulatory functions through its effect on the cyclic AMP: cyclic AMP-dependent protein kinase system.     

Correlation between changes in intracellular level of cyclic AMP, activation of cyclic AMP-dependent protein kinase, and the morphology of Chinese hamster ovary cells in culture.

The morphological conversion of Chinese hamster ovary cells induced by treatment with dibutyryl cyclic AMP is correlated with increases in the intracellular level of cyclic AMP and the activation of cyclic AMP-dependent protein kinase. When cholera toxin is used to induce the increase in intracellular cyclic AMP, a similar correlation is obtained. Treatment of cells with prostaglandin E₁, which causes a transient increase in intracellular cyclic AMP and a transient activation of protein kinase activity, does not result in the morphology change. From these studies we conclude that a stable activation of the cyclic AMP-dependent protein kinase, which results from an increase in intracellular cyclic AMP, induces the morphological conversion of Chinese hamster ovary cells through phosphorylation of one or more cellular components.     

Acetylcholinesterase molecular forms from rat and human erythrocyte membrane

Summary Inhibition of growth of PY815 mouse mastocytoma cells in vitro by N⁶, O²-dibutyryladenosine 3,5 cyclic monophosphate (DB cyclic AMP) was accompanied by increases in intracellular cyclic AMP and histamine and minor changes in cytosolic cyclic AMP-dependent histone kinase activity. However, DEAE-cellulose chromatography revealed substantial changes in the relative proportions of the principal cyclic AMP-dependent protein kinases and in free cyclic AMP-binding protein after DB cyclic AMP treatment. The activity of cytosolic cyclic AMP-dependent protein kinase type I (PK_I) decreased relative to cyclic AMP-dependent protein kinase type II (PK_II) and there was an increase in a cytosol cyclic AMP-binding protein with little associated protein kinase activity. The relative changes in activity of PK_I, PK_II and cyclic AMP binding protein after DB cyclic AMP treatment may reflect events important in the regulation of growth and differentiation of mast cells.Abbreviations DB cyclic AMP N⁶,O²-dibutyryladenosine-3, 5-cyclic monophosphate - cyclic AMP adenosine 3,5-cyclic monophosphate - PK_I type I cyclic AMP-dependent protein kinase - PK_II type II cyclic AMP-dependent protein kinase     

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.     

Properties and cellular distribution of cyclic AMP-dependent protein kinase from thymus

A protein kinase that catalyzes the phosphorylation of histone was partially purified from rat thymus, and the rate of histone phosphorylation was stimulated three- to fourfold by 1 × 10^?6 M adenosine 3′,5′-monophosphate (cyclic AMP). Thymic protein kinase was more active than the enzyme from spleen. Histone fractions f1, f2a, f2b, and f3 were all capable of serving as phosphate acceptors for the thymic protein kinase, and the rate of phosphorylation of each fraction was stimulated by cyclic AMP. The ability of various 3′,5′-mononucleotides to stimulate protein kinase activity was compared. Inosine 3′,5′-monophosphate (cyclic IMP) was the most effective substitute for cyclic AMP. The cellular distribution of cyclic AMP-dependent protein kinase and adenylate cyclase activities in the thymus was determined. Cyclic AMP-dependent protein kinase activity is present in both small thymocytes and residual thymic tissue. The specific activity of protein kinase from residual tissue, both for basal and cyclic AMP-stimulated enzyme, was greater than that of enzyme from small thymocytes. In contrast to this, adenylate cyclase activity is predominately localized in the thymocytes.     

Cyclic AMP-dependent protein kinase from Ustilago maydis

Summary Ustilago maydis was surveyed for cyclic AMP-dependent protein kinase activity. Using a combination of ion-exchange and molecular filtration techniques, we demonstrate that there is only one form of cyclic AMP-dependent protein kinase in the cytosolic fraction of the fungus. The kinase activity is specifically activated by cyclic AMP and utilizes protamine and kemptide as substrates. Most, if not all, of the cyclic AMP binding detected in the soluble fraction is associated with the protein kinase activity. Cyclic AMP-dependent protein kinase is completely dissociated by cyclic AMP into catalytic and regulatory subunits having an apparent molecular weight of 35 000 daltons as judged by sucrose gradient centrifugation.Post graduate fellow from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).Career investigator from CONICET.