Heat-stable Protein Kinase Inhibitor Research Articles

Activity ratio measurements reflect intracellular activation of adenosine 3',5'-monophosphate-dependent protein kinase in osteoblasts.

Parathyroid hormone, prostaglandin E2, and prostacyclin activate cAMP-dependent protein kinase in osteoblast-rich normal rat calvarial cells and in clonal rat osteogenic sarcoma cells of osteoblastic phenotype. The present study was undertaken to determine the activation of the enzyme in relation to cellular cAMP concentrations at increasing doses of the three hormones and also to test that the activity ratio measurement of the enzyme (ratio of the activity in the absence of cAMP to the activity in the presence of excess cAMP) was a true reflection of intracellular activation of the enzyme. With each hormone, using either normal or malignant osteoblasts, activation of the enzyme took place at hormone concentrations lower than those required to produce detectable changes in cAMP concentrations in the incubations. Stimulation of activity was abolished by addition of the heat-stable inhibitor of cAMP-dependent protein kinase, indicating that activation was of cAMP-dependent protein kinase alone. To demonstrate that protein kinase activation occurred intracellularly and not during sample preparation, charcoal was added at the time of cell disruption to absorb free cAMP. Under these conditions, no change was observed in the concentration of bovine parathyroid hormone required to cause activation of cAMP-dependent protein kinase. Finally, addition of purified cAMP-dependent protein kinase type I or type II to treated cells at the time of lysis did not result in significant activation of added isoenzyme, except at hormone concentrations sufficient to increase the total cAMP concentration of incubations. It is concluded that activity ratio measurement reflects the intracellular state of activation of cAMP-dependent protein kinase in the osteoblast-like cells treated by hormones and, furthermore, that only a fraction of the maximally generated cAMP is necessary for full enzyme activation.

Phosphorylation of glycogen synthase by cyclic AMP-independent casein kinase-2 from rabbit skeletal muscle.

Cyclic AMP-independent casein kinase-2 from rabbit skeletal muscle has been extensively purified by procedures including phosphocellulose, Bio-Gel A-1.5m, casein-Sepharose, and DEAE-cellulose column chromatographies. The casein kinase and glycogen synthase kinase activities are co-purified throughout the purification. The casein kinase-2 has Mr approximately equal to 135,000 as determined by glycerol density gradient centrifugation and by gel filtration. Analysis of the purified kinase by gel electrophoresis in the presence of sodium dodecyl sulfate reveals the presence of two major protein bands having Mr = 42,000 and 27,000 in a ratio of 0.85:1. The kinase phosphorylates glycogen synthase, casein, and phosvitin either in the presence of ATP or GTP; however, the Km values for GTP are slightly higher than those of ATP. The activity of this kinase is inhibited by heparin but is not affected by the addition of cyclic AMP, heat-stable inhibitor of cyclic AMP-dependent protein kinase, Ca2+, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or the combination of Ca2+ and calmodulin. The phosphorylation of the synthase by the kinase results in the incorporation of approximately 0.8 mol of PO4/subunit and a reduction in the synthase activity ratio from 0.82 to 0.6. The extent of phosphorylation of the synthase catalyzed by casein kinase-2 is similar to that by phosphorylase kinase; however, the sites phosphorylated by these two kinases are different. The relationships of casein kinase-2 to the cyclic AMP-independent synthase kinases that have been described by other laboratories and to the casein kinases isolated from other tissues are discussed.

Characterization of endogenous protein phosphorylation in isolated rat liver lysosomes.

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.

Microsomal cAMP-independent histone H1 kinase activity in plasmacytoma, morris hepatoma and normal liver

A protein kinase activity with high specificity for histone H1 was isolated from mouse plasmacytoma, Morris hepatoma and normal mouse liver and compared by ion exchange chromatography after DEAE-cellulose, hydroxylapatite and Sephadex G-200 chromatography. This cAMP-independent histone H1 kinase is not affected by the heat-stable cAMP-dependent protein kinase inhibitor. It has the following particular properties: it prefers GTP to ATP as substrate and was found to be present with a great activity only in neoplastic tissues. No phosphatase activity was detected in the partially purified histone H1 kinase fraction from normal and neoplastic cells. These results suggest either an increase amount of histone H1 kinase and/or of its activator in neoplastic cells, or the presence of a strong inhibitor in normal cells. This histone H1 kinase appears to be analogous to the chromatin bound kinase which phosphorylates histone H1 at the NH2 and COOH terminal regions. We might suggest an implication of this kinase in the regulation of cell division.

Selective expression of type I and type II cyclic AMP-dependent protein kinases in subcellular fractions of concanavalin A-stimulated rat thymocytes

Total protein kinase activity and the expression of the type I and type II cyclic adenosine 3′:5′-monophosphate-dependent protein kinases were studied in subcellular fractions of rat thymocytes and the effect of concanavalin A treatment on protein kinase activity was assessed. At a concentration of 100 μ/ml of concanavalin A a marked decline of total nuclear protein kinase activity occurred which lasted approximately 20 to 90 min. Concomitantly, a twofold increase of total protein kinase activity in the 900 g supernatant fraction was observed which lasted from 5 to 30 min. Studies using the heat-stable protein kinase inhibitor revealed that the concanavalin A-mediated activity changes were primarily due to changes of cAMP-dependent protein kinase activity, whereas cAMP-independent protein kinase activity remained unchanged. Analysis of the type I and type II cAMP-dependent protein kinase isozyme pattern before and after concanavalin A treatment revealed a selective change of the relative expression of isozyme activities. Whereas type I protein kinase was the major nuclear isozyme before concanavalin A treatment, nuclear type II cAMP-dependent protein kinase increased markedly with a concomitant loss of type I isozyme expression. In the 900 g supernatant fraction, containing primarily the type II isozyme in unstimulated cells, concanavalin A treatment caused an increase of the expression of the type I isozyme. The concanavalin A-mediated relative changes of cAMP-dependent protein kinase isozyme expression were confirmed by photoaffinity labeling of the regulatory subunits R I and R II before and after concanavalin A stimulation. The intracellular concanavalin A-mediated isozyme changes were time dependent, exhibiting maximal effects about 20 min after concanavalin A addition. These results indicate that selective regulation of intracellular cAMP-dependent protein kinase isozyme expression may be a mechanism related to isozyme-specific phosphorylation of specific intracellular substrates in concanavalin A-activated thymocytes.

Regulation of 6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation.

Addition of glucagon to isolated rat hepatocytes resulted in inhibition of 6-phosphofructo-2-kinase (ATP:D-fructose-6-phosphate-2-phosphotransferase) activity in extracts of the cells and in a decrease in the intracellular level of fructose 2,6-bisphosphate. The effect on 6-phosphofructo-2-kinase was characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. To investigate the mechanism of action of glucagon, 6-phosphofructo-2-kinase from rat liver was partially purified by polyethylene glycol precipitation, DEAE-cellulose chromatography, (NH4)2SO4 fractionation, Sephacryl S-200 gel filtration, DEAE-Sephadex chromatography, and Sephadex G-100 gel filtration. Incubation of the purified enzyme with the catalytic subunit of the cyclic AMP-dependent protein kinase from rat liver and [gamma-32P]ATP resulted in 32P incorporation into a protein with a subunit Mr of 49,000 as determined by NaDodSO4 disc gel electrophoresis. Associated with this phosphorylation was an inhibition of 6-phosphofructo-2-kinase activity that was also characterized by a decrease in the affinity of the enzyme for fructose-6-phosphate. Both the phosphorylation and the inhibition of the purified 6-phosphofructo-2-kinase were blocked by addition of the heat-stable protein kinase inhibitor. It is concluded that the glucagon-induced decrease in fructose 2,6-bisphosphate levels observed in isolated hepatocytes is due, at least in part, to cyclic AMP-dependent phosphorylation and inhibition of 6-phosphofructo-2-kinase.

Hormone-induced phosphorylation of a 16000 Dalton polypeptide following meiosis reinitiation in starfish oocytes

In vitro phosphorylation of endogenous proteins is increased in homogenates prepared from 1-methyladenine-treated starfish oocytes when compared with control oocytes, although addition of the hormone to homogenates from control oocytes has no such effect. Following hormonal stimulation the best endogenous substrate is by far a 16 000 dalton (D) protein, the content of which also seems to increase, perhaps through proteolysis of a 21 000 D precursor. cAMP-dependent protein kinases are not involved in either basal or hormone-stimulated phosphorylations, as demonstrated by the lack of effect of either cAMP or of the heat-stable inhibitor of cAMP-dependent protein kinase on the extent of phosphorylation of individual endogenous substrates. Addition of 0.1 mM Ca 2+ decreases to some extent the protein kinase activity in starfish homogenates and specifically suppresses the phosphorylation of a 40 000 D membrane protein. Starfish oocytes appear to contain myosin light chain kinase activity, as shown by the ability of homogenates to catalyse phosphorylation of exogenous 20 000 D myosin light chains.

Purification and characterization of a specific histone H1 protein kinase from mouse plasmacytoma.

A protein kinase with high specificity for histone H1 was purified from a plasmacytoma microsomal fraction by a high-salt wash, ammonium sulfate precipitation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-200 columns, and the main properties of this kinase were studied. A sulfhydryl compound, such as 2-mercaptoethanol or dithiothreitol, was necessary for full activity. The optimum pH was 7.4-7.8. After purification, the histone H1 kinase was not stimulated by cAMP or cGMP. It was not inhibited by the heat-stable cAMP-dependent protein kinase inhibitor from beef heart. It utilized preferentially GTP over ATP as phosphate donor. Km values for ATP and GTP were 58 microM and 1.4 microM respectively; the Km for histone H1 was 14 microgram ml-1. The molecular weight was approximately 90 000 by gel-exclusion chromatography. Analysis of the purified H1-specific protein kinase by polyacrylamide gel electrophoresis in dodecylsulfate showed two bands having molecular weights of approximately 64 000 and 54 000. Many characteristics of this kinase were similar to those of the chromatin-bound protein kinase reported by other workers in rapidly proliferating cells.

Regulation of guinea pig heart phosphorylase kinase by cAMP, protein kinase, and calcium.

In skeletal muscle the activation of phosphorylase kinase (PK) associated with phosphorylation of the enzyme can be measured as an increase in the pH 6.8:8.2 activity ratio. Phosphorylation leads to a 20- to 30-fold increase in PK activity (PKA) at pH 6.8 and a large decrease in the Km for phosphorylase. Perfused guinea pig hearts exposed to isoproterenol (0.3 microM) showed an increase in PKA, but without an increase in the pH 6.8:8.2 activity ratio. In a 10-fold dilution of guinea pig heart cytosol exposed to cAMP + methylisobutylxanthine, PKA was stimulated twofold at pH 7.5. Addition of exogenous protein kinase stimulated PKA fourfold. Both methods of activation were reversible and were blocked by the heat-stable inhibitor of protein kinase. Guinea pig heart PK was Ca2+-dependent requiring 0.6 microM Ca2+ for half-maximal activity. Kinetic studies indicate that the Km of guinea pig heart PK for phosphorylase b at pH 6.8 is not markedly reduced after in vitro activation (35%). This could explain the observed lack of increase in the pH 6.8:8.2 activity ratio after exposure of hearts to isoproterenol. The time course for the activation of inotropic state and the glycogenolytic pathway in perfused guinea pig hearts by isoproterenol showed that these processes were maximally activated within 25 s. However, PK remained activated for 2 min, long after the other biochemical and physiological parameters had returned to control values. These data suggest that Ca2+, not phosphorylation state, is important in regulating the return of dP/dt to control levels after beta-adrenergic stimulation.

Effect of hypothyroid status on adenosine 3',5'-mono-phosphate-dependent protein kinase of skeletal, heart and diaphragm muscle of rats.

Properties of cyclic AMP-dependent protein kinases from skeletal, heart and diaphragm muscles of hypothyroid rats were compared. Increased enzyme activity was observed in skeletal muscle from hypothyroid rats after DEAE-cellulose chromatography. Changes in isozyme distribution were also shown in the hypothyroid status. The elution profile on DEAE-cellulose suggested a possible translocation of the enzyme from the particulate to the soluble fraction in the heart of hypothyroid rats. The turnover rate of the enzyme protein decreased in the skeletal muscle of hypothyroid rats, but the other two organs showed no change even in the hypothyroid status. The activity of heat-stable protein kinase inhibitor increased in the skeletal and the diaphragm muscles in hypothyroid rats, whereas the activity in the heart decreased in the hypothyroid status. These data suggested the possibility that changes in enzyme properties, modification of isozyme distribution, and changes in modulator activity might account for the modulation of muscle function in hypothyroid rats.

EFFECT OF HYPOTHYROID STATUS ON ADENOSINE 3′,5′-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE OF RAT KIDNEY

AbstractChanges in the properties of cyclic AM P-dependent protein kinase were studied in the kidney of hypothyroid rats. The activity of the crude extract was greater in hypothyroid rats than in normal ones. The ratio of type I isozyme to type II isozyme in hypothyroid rats was about twice that in the normal rats with enzymes from both the cortex and medulla. A higher affinity for the substrates was shown in the enzymes of the hypothyroid rat kidney. The effects of actinomycin D and of heat-stable protein kinase inhibitor on the enzyme activity were the same in both normal and hypothyroid rats. These changes in enzyme properties and isozyme distribution may well play an important role in the hypothyroid rat kidney.

EFFECT OF HYPOTHYROID STATUS ON ADENOSINE 3′,5′-MONO-PHOSPHATE-DEPENDENT PROTEIN KINASE OF SKELETAL, HEART AND DIAPHRAGM MUSCLE OF RATS

AbstractProperties of cyclic AMP-dependent protein kinases from skeletal, heart and diaphragm muscles of hypothyroid rats were compared. Increased enzyme activity was observed in skeletal muscle from hypothyroid rats after DEAE-cellulose chromatography. Changes in isozyme distribution were also shown in the hypothyroid status. The elution profile on DEAE-cellulose suggested a possible translocation of the enzyme from the particulate to the soluble fraction in the heart of hypothyroid rats. The turnover rate of the enzyme protein decreased in the skeletal muscle of hypothyroid rats, but the other two organs showed no change even in the hypothyroid status. The activity of heat-stable protein kinase inhibitor increased in the skeletal and the diaphragm muscles in hypothyroid rats, whereas the activity in the heart decreased in the hypothyroid status. These data suggested the possibility that changes in enzyme properties, modification of isozyme distribution, and changes in modulator activity might account for the modulation of muscle function in hypothyroid rats.

Effect of hypothyroid status on adenosine 3',5'-monophosphate-dependent protein kinase of rat kidney.

Changes in the properties of cyclic AMP-dependent protein kinase were studied in the kidney of hypothyroid rats. The activity of the crude extract was greater in hypothyroid rats than in normal ones. The ratio of type I isozyme to type II isozyme in hypothyroid rats was about twice that in the normal rats with enzymes from both the cortex and medulla. A higher affinity for the substrates was shown in the enzymes of the hypothyroid rat kidney. The effects of actinomycin D and of heat-stable protein kinase inhibitor on the enzyme activity were the same in both normal and hypothyroid rats. The changes in enzyme properties and isozyme distribution may well play an important role in the hypothyroid rat kidney.

Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcoplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg 2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol P i/mg protein were incorporated in 10 min at 30°C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25–100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occured on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca 2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca 2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.

Resensitization of the Desensitized Follicular Adenylyl Cyclase System to Luteinizing Hormone*

LH-induced desensitization of the adenylyl cyclase system in a cell-free membrane preparation from preovulatory porcine follicles exhibits a critical dependence upon Mg and ATP (1). The membrane-rich preparation was found to contain endogenous cAMP-dependent and cAMP-independent protein kinases as well as phosphorprotein phosphatases. Endogenous phosphatase activity was enchanced by by Mn2+ and dithiothreitol. The addition of either Mn2+ or dithiothreitol to the porcine follicular membrane preparation incubated under desensitizing conditions promoted a specific concentration-dependent reversal of the LH-induced desensitization of the adenylyl cyclase system. The addition of exogenous phosphoprotein phosphatase, partially purified from procine follicular cytosol, also reversed LH-induced desensitization in a concentration-dependent manner. Boiling of the phophatase preparation prevented reversal of desensitization. The addition of either exogenous beef heart cAMP-dependent protein kinase or heat-stable protein kinase inhibitor did not modify LH-induced desensitization of the follicular adenylyl cyclase system. These results provide indirect evidence that while LH-induced desensitization is not mediated by a cAMP-dependent protein kinase, reversal of desensitization can be promoted by activation of endogenous phosphatase and the addition of a homologous phosphatase preparation.

Protein Inhibitors of Dog-Liver Phosphorylase Phosphatase Dependent on and Independent of Protein Kinase

Dog liver extracts were shown to contain two kinds of heat-stable protein inhibitors of phosphorylase phosphatase. One type (inhibitor 1) depends upon phosphorylation by protein kinase for its inhibitory activity and can be reversibly inactivated by the action of a phosphatase. The other type (inhibitor 2) is spontaneously active and is not affected by protein kinase or phosphatase. The heat-stable phosphatase inhibitors are distinct from the heat-stable protein kinase inhibitor. Inhibitor 1 appears to be an asymmetrical molecule, characterized on gel filtration by a Stokes radius of 3.4 nm, and in sucrose-density-gradient centrifugation by an s20,w value of 1.6 S. Two forms of the spontaneously active inhibitor 2 (termed α and β) were observed which had different specificities towards different phosphatase preparations. Stokes radii of 4.3 nm and 2.7 nm were found for inhibitor 2α and 2β respectively, but sucrose-density-gradient centrifugation revealed the same s20,w value of 1.6 S. Fractions of high-molecular-weight phosphorylase phosphatase preparations from dog liver cytosol which did not contain measurable heat-stable protein inhibitors, could be dissociated and activated by treatment with ethanol or urea. This would suggest that the high-molecular-weight phosphatase, which is insensitive to the protein inhibitors, does not consist of a complex of catalytic unit(s) and these heat-stable inhibitor proteins.

Rabbit ovarian protein kinases. II. Effect of an ovulatory dose of human chorionic gonadotropin or luteinizing hormone on the multiplicity of follicular and luteal protein kinases.

DEAE-cellulose chromatography of the 105,000 X g supernatant fraction (cytosol) obtained from popped estrous rabbit follicles revealed the presence of a single form of cAMP-dependent protein kinase, designated protein kinase 3. The iv injection of an ovulatory dose of hCG to estrous rabbits promoted the appearance of a second, transient peak of cytosol cAMP-dependent protein kinase, protein kinase 1. Protein kinase 1 was detected within 10 min of hCG administration but had regressed to undetectable levels by 24 h in corpora lutea (CL) of pseudopregnancy and by 72 h in CL of pregnancy. Ovulation and subsequent CL formation were accompanied by the appearance of a third form of cAMP-dependent protein kinase, designated protein kinase 2. Protein kinase 2 was present within 2 h after hCG administration and persisted as a major form of cytosol cAMP-dependent protein kinase throughout the life span of CL. All three forms of protein kinase were inhibited by the heat-stable protein kinase inhibitor from rabbit skeletal muscle, possessed cAMP-binding activity, and were markedly stimulated by 10(-7) M cAMP. The activity of protein kinase 3 in CL of pregnancy, in corpora albicantia, and in interstitial tissue was markedly greater than that in follicles or in CL of pseudopregnancy, while the activity of protein kinase 2 remained relatively constant throughout the luteal life span. The iv injection of a luteolytic dose of hCG to 4-day pseudopregnant rabbits promoted no alterations of the protein kinase elution profile upon DEAE-cellulose chromatography of the luteal cytosol obtained 10 min to 3 days post-hCG injection. However, with dedifferentiation of corpora albicantia into interstitial tissue, the cAMP dependency of protein kinase 2 was reduced. The results indicate that the enzymatic activity and multiplicity of cAMP-dependent protein kinases in the cytosol of ovarian structures are subject to regulation by LH (hCG) and depend upon the various reproductive stages of the rabbit.

Rabbit Ovarian Protein Kinases. I. Effect of an Ovulatory Dose of Human Chorionic Gonadotropin or Luteinizing Hormone on the Subcellular Distribution of Follicular and Luteal Protein Kinases*

Studies on the subcellular distribution of protein kinase activity in popped estrous follicles from rabbit ovaries revealed that 15% of the total cellular protein kinase activity was compartmentalized in the nuclear, mitochondrial, and microsomal fractions. About 50% of the particulate protein kinase activity was unaffected by the heat-stable protein kinase inhibitor and was thus cAMP-independent. The majority of cellular protein kinase activity was identified in the 105,000 X g supernatant fraction as cAMP-dependent. hCG- or coital-induced ovulation and subsequent corpus luteum (CL) formation, and hCG-induced luteal regression promoted changes and a redistribution of protein kinase activity among the subcellular fractions. In follicles, hCG promoted a transient decline of nuclear protein kinase activity as well as transient increases of the relative amount of protein kinases localized in the microsomal fractions before ovulation. In CL induced by a fertile mating, the specific activity as well as the total amount of protein kinases in the nuclear fraction were reduced 2-fold. Mitochondrial protein kinase activity from CL of pseudopregnancy and pregnancy was reduced 2-fold. The relative amount of protein kinase activity in microsomes of CL was increased 2-fold, but the specific activity was not affected. hCG-induced luteal regression resulted in a transient decline of the nuclear protein kinase activity in CL of 4-day pseudopregnant rabbits. In interstitial tissue, the specific activity of the nuclear protein kinase was increased over luteal levels, the mitochondrial-specific protein kinase remained at the reduced luteal levels, and the microsomal and cytosol protein kinase specific activities increased 2-fold. Studies with the heat-stable protein kinase inhibitor revealed that the hCG- or coital-induced redistribution of intracellular protein kinase affected both the cAMP-dependent and cAMP-independent activity to a similar degree and no changes of the relative distribution of cAMP-dependent vs. cAMP-independent activity were observed. These results indicate that the intracellular distribution and enzymatic activity of cAMP-dependent protein kinases in ovarian structures are subject to regulation by LH (hCG) and depend upon the various reproductive stages of the rabbit.

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L 6 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 L 6 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 f 2b, 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

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.