Calmodulin intracelluar concentration and cAMP-dependent protein kinase activity in human sperm samples in relation to sperm motphlogy and motlity

Calmodulin concentration and cAMP-dependent protein kinase activity have been determined in human sperm samples. No significant differences have been noticed in the motility index of two groups of sperm samples differing in their intracellular concentration of calmodulin. It was however found that a low intracellular clamodulin concentration is frequently associated with particular anomalies of the head and tail region. In contrast, a positive correlation has been demonstrated between the motility index and the whole extractable cAMP-dependent protein activity of the different sperm samples. In suspension s, When the cAMP-dependent protein kinase activity was measured on intact sperm suspensions, a positive correlation between the activity and the motility index was also found.     

Changes in calmodulin level and cAMP-dependent protein kinase activity during epididymal maturation of ram spermatozoa

Calmodulin concentration and cAMP-dependent protein kinase activity were simultaneously determined on ram spermatozoa collected by cannulation of successive segments of the epididymal tubule. Epididymal transit was characterized on one hand by an overall decrease in the calmodulin level and on the other by a dramatic rise in the cAMP-dependent protein kinase activity. In contrast to the calmodulin level, the cAMP-dependent protein kinase activity was correlated with the acquisition of flagellar beat. No further alterations in the level of these two proteins could be detected as spermatozoa acquired progressive motility.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Evolution of Ca2+- and cAMP-dependent regulatory mechanisms during ram spermatogenesis

Calmodulin level and cAMP-dependent protein kinase activity of ram germ cells at different stages of spermatogenesis have been determined. Calmodulin levels decrease during maturation. Simultaneously, calmodulin localization changes during cell differentiation. In round, elongating, and elongated spermatids, calmodulin is closely associated with the developing acrosome; in spermatozoa, it becomes present in the postacrosome, the neck region and the tail. Protein kinase activity is relatively low in testicular cells but increases dramatically during epididymal maturation of spermatozoa. A concerted regulation by cAMP and Ca2+ of biochemical events in spermatogenic cells and spermatozoa is suggested.     

beta-Adrenergic agonists increase phosphorylation of elongation factor 2 in cardiomyocytes without eliciting calcium-independent eEF2 kinase activity

The beta-adrenergic agonist isoproterenol increased the phosphorylation of elongation factor eEF2 in ventricular cardiomyocytes from adult rats (ARVC). Phosphorylation of eEF2 inhibits its activity, and protein synthesis was inhibited in ARVC concomitantly with increased eEF2 phosphorylation. eEF2 kinase activity in ARVC extracts was completely dependent upon Ca(2+)/calmodulin. In contrast to other cell types, however, treatments designed to raise intracellular cAMP failed to induce Ca(2+)/calmodulin-independent activity. Instead, they increased maximal eEF2 kinase activity. Similar data were obtained when partially purified ARVC eEF2 kinase was treated with cAMP-dependent protein kinase in vitro. These data suggest that ARVC possess a distinct isoform of eEF2 kinase.     

Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites

Smooth muscle myosin light chain kinase is phosphorylated in vitro by protein kinase C purified from human platelets. When myosin light chain kinase which has calmodulin bound is phosphorylated by protein kinase C, 0.8-1.1 mol of phosphate is incorporated per mol of myosin light chain kinase with no effect on its enzyme activity. Phosphorylation of myosin light chain kinase with no calmodulin bound results in the incorporation of 2-2.4 mol of phosphate and significantly decreases the rate of myosin light chain kinase activity. The decrease in myosin light chain kinase activity is due to a 3.3-fold increase in the concentration of calmodulin necessary for the half-maximal activation of myosin light chain kinase. The sites phosphorylated by protein kinase C and the catalytic subunit of cAMP-dependent protein kinase were compared by two-dimensional peptide mapping following extensive tryptic digestion of phosphorylated myosin light chain kinase. The single site phosphorylated by protein kinase C when calmodulin is bound to myosin light chain kinase (site 3) is different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 1). The additional site that is phosphorylated by protein kinase C when calmodulin is not bound appears to be the same site phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 2). These studies confirm the important role of site 2 in binding calmodulin to myosin light chain kinase. Sequential studies using both protein kinase C and the catalytic subunit of cAMP-dependent protein kinase suggest that the phosphorylation of site 1 also plays a part in decreasing the affinity of myosin light chain kinase for calmodulin.     

cAMP, calmodulin-dependent stimulation and stability to proteolysis of Ca 2+ transport in the heart sarcoplasmic reticulum

The calmodulin content in cardiomyocyte cytosol of hypoxic myocardium is increased compared to normal level. This is unaccompanied by differences in the stimulating effect of calmodulin on Ca2+ transport in sarcoplasmic reticulum (SR) of ischemic heart. The decrease of the endogenous cAMP-dependent protein kinase activity in ischemia is associated with the lowered resistance to trypsinolysis of Ca2+ transport in SR (trypsin/microsomal protein ratio is 1:10) with simultaneous Ca-ATPase activation. In the presence of exogenous protein kinase and cAMP the protective effect of phosphorylation on Ca2+ transport in SR vesicles of hypoxic cardiomyocytes treated with trypsin for 10 min reaches the same level as in intact heart.     

Interaction of calmodulin with myosin light chain kinase and cAMP-dependent protein kinase in bovine brain

Myosin light chain kinase and a fraction of type II cAMP-dependent protein kinase have been partially purified from bovine brain by affinity chromatography on calmodulin-Sepharose. The myosin kinase was purified approximately 3700-fold and has an estimated molecular weight of 130,000 +/- 10,000 by sodium dodecyl sulfate gel electrophoresis. A fraction of soluble cAMP-dependent protein kinase also bound to calmodulin-Sepharose and was purified 2300-fold. A fraction of this cAMP-dependent protein kinase after purification by glycerol gradient centrifugation was shown to contain the two subunits of calcineurin, a major calmodulin-binding protein in brain, and the two subunits of type II cAMP-dependent protein kinase in a ratio of 1:1:2:2. Its sedimentation coefficient was 8.1 S and 9.0 S when centrifuged in the absence or presence of calmodulin, suggesting the formation of a complex between calmodulin and protein kinase. Our results suggest the possibility that calcineurin may be involved in the interaction between the protein kinase and calmodulin. Furthermore, our studies imply that the regulatory subunit of the cAMP-dependent protein kinase, but not the catalytic subunit, is the site of interaction with calmodulin since the catalytic subunit of protein kinase was partially resolved from the complex by cAMP.     

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.     

Prostacyclin-induced activation of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase in rat antral and fundic gastric mucosa.

The present investigations showed that after oral prostacyclin administration (100 micrograms/kg) as soon as the intracellular level of cAMP is elevated the activation of cAMP-dependent protein kinase follows in both parts (antrum and fundus) of rat gastric mucosa. The enzyme activation seems to be more significant in the fundic region which is in a complete agreement with the previously published results, i.e. the fundic mucosa reacts with de novo protein synthesis toward noxious agents (resulting finally in new cell formation), while the antral mucosa is more durable against damaging noxae. Taking into consideration all available data in the literature it seems that the intracellular effect of the exogenously administered prostacyclin in the gastric mucosa is a polyphasic effect, which contains the following consecutive steps: 1. Binding to the cell surface; 2. Effect on the intracellular second messenger system, (cAMP, cGMP); 3. Activation of the calmodulin system; 4. cAMP-dependent protein kinase activation; 5. DNA, RNA changes; 6. Influence on protein synthesis, and finally; 7. New cell formation.     

Regulation of human platelet membrane Ca2+ transport by cAMP- and calmodulin-dependent phosphorylation

We have examined the effects of added cAMP-dependent protein kinase and endogenous calmodulin-dependent kinase on Ca2+ transport in purified internal membranes from human platelets. Both Ca2+ uptake and Ca2+-ATPase activity were maximally stimulated about 2-fold by addition of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase inhibitor reduced both Ca2+ uptake and Ca2+-ATPase activities at concentrations which also inhibited cAMP-dependent protein phosphorylation. In addition, concerted stimulation of Ca2+-ATPase by exogenous calmodulin and added catalytic subunit of cAMP-dependent protein kinase was observed. A 22-kDa protein was phosphorylated by both cAMP-dependent and calmodulin-dependent kinases at the same rate as stimulation of the Ca2+-ATPase. Cyclic AMP-dependent phosphorylation of the 22-kDa polypeptide was inhibited by the protein kinase inhibitor and calmodulin-dependent phosphorylation was inhibited by chlorpromazine and EGTA. These results are consistent with the hypothesis that one mode of control of Ca2+ homeostasis in platelets may be similar to the phospholamban system in cardiac muscle.     

Effect of chronic electrostimulation of rabbit skeletal muscle on calmodulin level and protein kinase activity

(a) Chronic electrostimulation of fast-twitch skeletal muscles makes them resemble slow-twitch muscles. The involvement of second-messenger cascades in this muscle reprogramming is not well understood. The goal of this study was to examine protein kinase activities and calmodulin levels as a function of the duration of electrostimulation. (b) Fast-twitch rabbit muscle was subjected to continuous low-frequency electrostimulation for 2 weeks. The extensor digitorum longus was taken and examined for calmodulin concentration and cAMP-dependent (PKA). Ca(2+)-phospholipid-dependent (PKC) and Ca(2+)-calmodulin-dependent (CaM kinase or PKB) protein kinase activities. (c) Electrostimulation for 14 days led to a significant increase in total calmodulin level and PKB activity, both rising in the cytosolic fraction. Protein kinase C translocated to the membrane fraction, although total activity did not change. (d) These changes could be related with electrostimulation-induced changes in excitation-contraction coupling.     

Activities of cAMP-dependent protein kinase and enzymes of 2′,5′-oligoadenylate metabolism in NIH 3T3 cells deepening into the resting state

The activity of cAMP-dependent protein kinase was found to increase continuously in the NIH 3T3 cells, deepening into the resting state. The increase correlated with intracellular level of heat-stable protein inhibitor of the protein kinase rather than with the cAMP content. The elevation of 2',5'-oligo(A) synthetase activity and the decrease in 2'-phosphodiesterase activity were also observed in the cells sinking into the resting state. The variations in enzyme activities were similar to those caused by the increase in the intracellular cAMP content described elsewhere. These results agree with the idea that the cAMP-dependent protein kinase is involved in the regulation of the enzymes of 2',5'-oligo(A) metabolism.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid-dependent protein kinase

Protein kinase C incorporates phosphate into two sites of myosin light chain kinase (MLC-kinase) in the absence of calmodulin. Phosphorylation is all but abolished in the presence of Ca2+ and calmodulin, suggesting that both sites of phosphorylation are close to the calmodulin binding site. The phosphorylation of MLC-kinase results in an approximately 10-fold increase in the dissociation constant of MLC-kinase for calmodulin. Following phosphorylation (2 mol/mol of enzyme) of MLC-kinase by protein kinase C, an additional 2 mol of phosphate can be incorporated into the MLC-kinase apoenzyme by the cAMP-dependent protein kinase. Different maps of phosphopeptides were obtained by tryptic hydrolysis from MLC-kinase preparations phosphorylated by each kinase. The phosphorylation sites for the cAMP-dependent kinase were located in a fragment of approximately 25,000 daltons. In contrast the phosphorylation sites for protein kinase C are found in a much smaller tryptic peptide. These results suggest that the phosphorylation sites on MLC-kinase are different for protein kinase C and for cAMP-dependent protein kinase. However, phosphorylation in both regions results in a reduced affinity for calmodulin.     

Association of calmodulin with peptide analogues of the inhibitory region of the heat-stable protein inhibitor of adenosine cyclic 3',5'-phosphate dependent protein kinase

A 20-residue peptide analogue (IASGRTGRRNAIHDILVSSA) of the 8000-dalton heat-stable cAMP-dependent protein kinase inhibitor undergoes efficient calcium-dependent binding by calmodulin, with Kd approximately 70 nM when calcium is present. It is a potent inhibitor of smooth muscle myosin light chain kinase and of the calmodulin-dependent phosphatase activity of calcineurin. At concentrations above 3 microM, the peptide stimulates the basal activity of calcineurin. The native protein kinase inhibitor has no effect on the catalytic activity of myosin light chain kinase and is moderately inhibitory to both the calmodulin-dependent and -independent phosphatase activity of calcineurin. Competition experiments using excess concentrations of calcineurin and calmodulin suggest that the primary interaction of the native heat-stable inhibitor is with the catalytic subunit of protein kinase. Dansylcalmodulin exhibits only a weak interaction with the inhibitor. Observations on deletion peptides of the 20-residue analogue help to delineate the overlapping peptide binding specificities of the cAMP-dependent protein kinase [Scott, J. D., Glaccum, M. B., Fischer, E. H., & Krebs, E. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1613-1616] and calmodulin. In both cases, the most effectively bound peptides contain the RTGRR sequence.     

The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets.

Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.     

Characterization of calmodulin-activated protein kinase activity of rat adipocyte endoplasmic reticulum fraction

Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function.     

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 900g 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 900g 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.     

Inhibitors of IL-2 production and IL-2 receptor expression in human leukemic T-cell line, Jurkat

1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, suppressed interleukin 2 (IL-2) production and IL-2 receptor (IL-2R) expression of the human leukemic T-cell line, Jurkat, induced by 12-O-tetradecanoyl-phorbol-13-acetate and phytohemagglutinin-P. This effect was significant at 5 microM H-7 without loss of cell viability. Such activity was not observed with N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), a potent inhibitor of cGMP- and cAMP-dependent kinases, and a weak inhibitor of Ca2+-phospholipid-dependent protein kinase (protein kinase C). These findings suggest that protein kinase C is more closely associated with IL-2 receptor expression and IL-2 production of T cells than cGMP- or cAMP-dependent kinases. In addition, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, suppressed both IL-2 production and IL-2R expression. Cycrosporin A (Cy A), a potent immunosuppressive drug, markedly inhibited IL-2 production of Jurkat cells whereas it did not affect the IL-2R expression. Thus, the mechanism of action of Cy A appears to differ from that of the protein kinase inhibitor, H-7, and the calmodulin inhibitor, W-7.