Characterization of a calcium and diacylglycerol-activated and phospholipid-dependent protein kinase in the pupal brain of the tobacco hornworm,Manduca sexta: The cytosolic enzyme

A calcium and diacylglycerol-activated and phospholipid-dependent protein kinase (protein kinase C; PKC) in the cytosol of the pupal brain of the tobacco hornworm Manduca sexta has been characterized. Two peaks of PKC activity were separated by DEAE-cellulose chromatography, both of which were dependent upon Ca²⁺, phosphatidylserine and 1,2 diolein. Maximal enzyme activity was obtained in the presence of 0.7 mM Ca²⁺ and 200 μg/ml of phosphatidylserine. Diacylglycerol (1,2 diolein; 50 μg/ml) enhanced PKC activity and calcium sensitivity markedly in the presence of phospholipids. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate substituted for diacylglycerol in the activation of PKC. By utilizing the differential inhibition of PKC and cyclic AMP-dependent protein kinase by trifluoperazine and protein kinase inhibitor, both enzymes were measured accurately in dilute, crude cytosol preparations using the common substrate histone H1.     

Phosphatidylserine affects specificity of protein kinase C substrate phosphorylation and autophosphorylation

Protein kinase C substrate phosphorylation and autophosphorylation are differentially modulated by the phosphatidylserine concentration in model membranes. Both substrate phosphorylation and auto-phosphorylation display a cooperative dependence on phosphatidylserine in sonicated vesicles composed of diacylglycerol and either phosphatidylcholine or a mixture of cell lipids (cholesterol, sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine). However, the concentration of phosphatidylserine required to support phosphorylation varies with individual substrates. In general, autophosphorylation is favored at intermediate phosphatidylserine concentrations, while substrate phosphorylation dominates at high phosphatidylserine concentrations. These different phosphatidylserine dependencies may reflect different affinities of particular substrates for negatively charged membranes. Increasing the negative surface charge of sonicated vesicles increases the rate of substrate phosphorylation. In contrast to the modulation exerted by phosphatidylserine, diacylglycerol activates protein kinase C equally toward substrate phosphorylation and autophosphorylation. These results indicate that both diacylglycerol and phosphatidylserine regulate protein kinase C activity in the membrane: diacylglycerol turns the enzyme on, while phosphatidylserine affects the specificity toward different substrates.     

Phosphorylation of different sites of acetyl CoA carboxylase by ATP-citrate lyase kinase and cyclic AMP-dependent protein kinase

Native acetyl CoA carboxylase was phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase and ATP-citrate lyase kinase to 1 and 0.5 mol/subunit respectively. Both protein kinases added together increased acetyl CoA carboxylase phosphorylation additively. Partial proteolysis of 32P-acetyl CoA carboxylase followed by electrophoretic analysis showed that the 32P-phosphopeptides generated from acetyl CoA carboxylase phosphorylated with lyase kinase were different from the peptides obtained from the enzyme phosphorylated by cyclic AMP-dependent protein kinase. Mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography showed that the major phosphopeptides phosphorylated by ATP-citrate lyase kinase were different from the major phosphopeptides phosphorylated by cyclic AMP-dependent protein kinase. The results suggest that at least one different site on acetyl CoA carboxylase is preferentially phosphorylated by each protein kinase.     

Phosphorylation of pig brain diacylglycerol kinase by endogenous protein kinase

Pig brain diacylglycerol kinase did not catalyze autophosphorylation. However, the kinase was phosphorylated on serine, when immunoprecipitated from the partially purified enzyme preparation preincubated with Mg2+ and [gamma-32P]ATP. The action of the endogenous protein kinase phosphorylating diacylglycerol kinase was independent of cyclic nucleotides and Ca2+, and became maximum at pH 5.5. Although the extent of enzyme phosphorylation was limited (maximally about 0.25 mol Pi incorporated per mol kinase), the results show that diacylglycerol kinase can be a phosphoprotein.     

Characterization of the sites phosphorylated on tyrosine hydroxylase by Ca2+ and phospholipid-dependent protein kinase, calmodulin-dependent multiprotein kinase and cyclic AMP-dependent protein kinase

Tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated rapidly by the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) purified from rat or sheep brain. Phosphorylation was stimulated 14-fold by Ca2+ and phosphatidylserine and occurred at a rate comparable with that of the phosphorylation of histone Hl. The phospholipid-dependent protein kinase phosphorylates a single site which is identical to that phosphorylated by cyclic AMP-dependent protein kinase and to the secondary site of phosphorylation by the calmodulin-dependent multiprotein kinase. The implications of these results with respect to the regulation of catecholamine biosynthesis in adrenal medulla are discussed.     

Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated stoichiometrically by either cyclic AMP-dependent protein kinase or calmodulin-dependent multiprotein kinase from skeletal muscle, but not by five other protein kinases tested. The activity of tyrosine hydroxylase was elevated 3-fold by cyclic AMP-dependent protein kinase, but no activation was observed after phosphorylation by calmodulin-dependent multiprotein kinase. Phosphorylation produced by cyclic AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase was additive, suggesting different sites of phosphorylation. This was confirmed by high-performance liquid chromatography analysis of tryptic phosphopeptides which demonstrated that the major sites phosphorylated by each protein kinase were distinct. A calmodulin-dependent multiprotein kinase that had identical properties and substrate specificity to the skeletal muscle enzyme was partially purified from rat pheochromocytoma. The possibility that this protein kinase is involved in the regulation of tyrosine hydroxylase activity in adrenergic tissue in vivo is discussed.     

The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase

We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.     

Calcium, phospholipid turnover and transmembrane signalling

Turnover of phosphatidylinositol, which is provoked by various neurotransmitters, peptide hormones and many other biologically active substances, appears to serve as a signal for the transmembrane control of protein phosphorylation through activation of a novel protein kinase (C-kinase). The activation of this enzyme absolutely requires Ca2+ and phosphatidylserine. Diacylglycerol derived from the receptor-linked breakdown of phosphatidylinositol dramatically increases the affinity of C-kinase for Ca2+, and thereby renders this enzyme fully active without a net increase in the concentration of Ca2+. Under appropriate conditions synthetic diacylglycerol directly added to intact cell systems activates C-kinase fully without interaction with surface receptors. By using such synthetic diacylglycerol and the Ca2+ ionophore A23187, it is shown that either receptor-linked protein phosphorylation or Ca2+ mobilization alone is merely a prerequisite but not a sufficient requirement, and both are synergistically effective for causing a full physiological cellular response. In some tissues cyclic nucleotides, both cyclic AMP and cyclic GMP, may inhibit the receptor-linked breakdown of phosphatidylinositol, and appear to provide negative control that prevents over-response.     

Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

The purpose of this investigation was to characterize the phosphorylation of bovine cardiac troponin by cyclic AMP-dependent protein kinase. The purified troponin-tropomyosin complex from beef heart contained 0.78 +/- 0.15 mol of phosphate per mol of protein. Analysis of the isolated protein components indicated that the endogenous phosphate was predominately in the inhibitory subunit (TN-I) and the tropomyosin-binding subunit (TN-T) of troponin. When cardiac troponin or the troponin-tropomyosin complex was incubated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, the rate of phosphorylation was stimulated by cyclic AMP and inhibited by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. The 32P was incorporated specifically into the TN-I subunit with a maximal incorporation of 1 mol of phosphate per mol of protein. The maximal amount of phosphate incorporated did not vary significantly between troponin preparations that contained low or high amounts of endogenous phosphate. The Vmax of the initial rates of phosphorylation with troponin or troponin-tropomyosin as substrates was 3.5-fold greater than the value obtained with unfractionated histones. The rate or extent of phosphorylation was not altered by actin in the presence or absence of Ca2+. The maximal rate of phosphorylation occurred between pH 8.5 and 9.0. At pH 6.0 and 7.0 the maximal rates of phosphorylation were 13 and 45% of that observed at pH 8.5, respectively. These results indicate that cyclic AMP formation in cardiac muscle may be associated with the rapid and specific phosphorylation of the TN-I subunit of troponin. The presence of endogenous phosphate in TN-T and TN-I suggests that kinases other than cyclic AMP-dependent protein kinase may also phosphorylate troponin in vivo.     

The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase

Acetyl-CoA carboxylase purified from isolated hepatocytes is activated dramatically by protein phosphatase treatment, concomitant with a reduction of the phosphate content from 3.7 to 1.1 mol/subunit. Glucagon treatment of the cells produces a further inactivation of the enzyme that is totally reversed by phosphatase treatment, and is associated with an increase in phosphate content of 0.8 mol/subunit, distributed in two peptides which contain the sites phosphorylated in vitro by the cyclic AMP-dependent and AMP-activated protein kinases. Sequencing of these peptides shows that the low activity of acetyl-CoA carboxylase is due to phosphorylation by the AMP-activated protein kinase, and not cyclic AMP-dependent protein kinase, even after glucagon treatment.     

Total conversion of glycogen synthase from the I- to the D-form by a cyclic AMP-independent protein kinase from rabbit skeletal muscle.

A newly discovered cyclic AMP-independent protein kinase, which catalyzes the total conversion of glycogen synthase from the I- to the D-form, has been isolated from rabbit skeletal muscle. This enzyme, designated glycogen synthase kinase, is separable from cyclic AMP-dependent protein kinase by column chromatography on phosphocellulose. Synthase kinase and cyclic AMP-dependent protein kinase are distinct in their specificity for protein substrates, the effects of cyclic AMP and the inhibitor of cyclic AMP-dependent protein kinase on their activities, and the extent to which they phosphorylate I-form glycogen synthase. The phosphorylation of I-form enzyme by synthase kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however only two of the phosphate sites seem predominantly to determine glucose-6-P dependence. The resulting multiply phosphorylated enzyme, which is highly dependent on glucose-6 P for activity, has a phosphate content comparable to the D-form enzyme isolated from rabbit muscle.     

Protein kinase C: Rapid enzyme purification and substrate-dependence of the diacylglycerol effect

Protein kinase C has been purified by a rapid method resulting in a high-yield, stable enzyme preparation. The catalytic and regulatory properties of this enzyme preparation were characterized employing histone H1 and HMG8, a proteolytic fragment of H1. The enzyme had a lower K_m for HMG8, and was stimulated more effectively by diacylglycerol and phorbol esters in the presence of this substrate. Furthermore, these activators markedly increased the K_m for HMG8 but not for H1. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate serine residues which are located in different, single tryptic peptides from HMG8.     

Amino acid sequence of the phosphorylation site of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was phosphorylated by cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Treatment of the 32P-labeled enzyme with thermolysin removed all of the radioactivity from the enzyme core and produced a single labeled peptide. The phosphopeptide was purified by ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography. The sequence of the 12-amino acid peptide was found to be Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser(P)-Ser-Ile-Pro-Gln. Correlation of the extent of phosphorylation with activity showed that a 50% decrease in the ratio of kinase activity to bisphosphate activity occurred when only 0.25 mol of phosphate was incorporated per mol of enzyme subunit, and maximal changes occurred with 0.7 mol incorporated. The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of the native bifunctional enzyme was compared with that of other rat liver protein substrates. The Km for 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (10 microM) was less than that for rat liver pyruvate kinase (39 microM), fructose-1,6-bisphosphatase (222 microM), and 6- phosphofructose -1-kinase (230 microM). Comparison of the initial rate of phosphorylation of a number of protein substrates of the cyclic AMP-dependent protein kinase revealed that only skeletal muscle phosphorylase kinase was phosphorylated more rapidly than the bifunctional enzyme. Skeletal muscle glycogen synthase, heart regulatory subunit of cyclic AMP-dependent protein kinase, and liver pyruvate kinase were phosphorylated at rates nearly equal to that of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, while phosphorylation of fructose-1,6-bisphosphatase and 6-phosphofructo-1-kinase was barely detectable. Phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was not catalyzed by any other protein kinase tested. These results are consistent with a primary role of the cyclic AMP-dependent protein kinase in regulation of the enzyme in intact liver.     

Calmodulin-dependent protein kinase II

Three multifunctional protein kinases, cyclic AMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II, are involved in signal transduction in response to their respective second messengers, cyclic AMP, diacylglycerol, and Ca2+. This review will summarize the key findings on calmodulin-dependent protein kinase II.     

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.     

Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit.

Cyclic GMP-dependent protein kinase was purified from foetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM respectively. The pH optimum for the enzyme activity ranged from 6 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP.     

Endogenous substrates for in vitro phosphorylation by cyclic AMP-dependent protein kinase from Dictyostelium discoideum

Endogenous proteins which could serve as substrates for cyclic AMP-dependent protein kinase in vitro were measured in cytosolic fractions at four stages of development. A peak of cyclic AMP-dependent phosphorylation occurred at the slug stage, coincident with the appearance of cyclic AMP-dependent protein kinase. After partial purification of the slug-stage extracts by DE-52 cellulose and Sephacryl S-300 chromatography, cyclic AMP dependency of six proteins was observed. The apparent subunit molecular weights of the proteins were greater than 200,000, 110,000, 107,000, 91,000, 75,000 and 69,000. Upon further purification of the cyclic AMP-dependent protein kinase by chromatofocusing, the endogenous substrates were separated from the enzyme. In addition, the enzyme separated into catalytic and regulatory subunits. If the purified catalytic subunit was added to heated S300 fractions, proteins with apparent molecular weights of 91,000 and 107,000 were specificity phosphorylated. The results show the stage-dependent appearance of a cyclic AMP-dependent protein kinase and point out several in vitro substrates for the enzyme.     

Protein kinase activities in rat pancreatic islets of Langerhans.

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.     

Polymyxin B is a more selective inhibitor for phospholipid-sensitive Ca2+-dependent protein kinase than for calmodulin-sensitive Ca2+-dependent protein kinase

Polymyxin B inhibited phospholipid-sensitive Ca²⁺-dependent protein kinase competitively with respect to phosphatidylserine (a phospholipid cofactor), with a K_i of 1.8 μM. It also inhibited myosin light chain kinase (a calmodulin-sensitive species of Ca²⁺-dependent protein kinase) competitively with respect to calmodulin, but with a higher K_i of 17.0 μM. Bacitracin, another polypeptide antibiotic, was much less active in inhibiting both enzymes. Polymyxin B and bacitracin were without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The findings indicate that polymyxin B, a surface active agent, effectively inhibited the phospholipid-sensitive enzyme presumably by interacting with phosphatidylserine.     

Regulation of human tyrosine hydroxylase activity. Effects of cyclic AMP-dependent protein kinase, calmodulin-dependent protein kinase II and polyanion

To determine the regulatory mechanism for human tyrosine hydroxylase, we examined modulations of the activity of the enzyme from human pheochromocytoma by cyclic AMP-dependent protein kinase, calmodulin-dependent protein kinase II and polyanion. The most remarkable activation was observed when the enzyme was assayed at physiological pH (pH 7) after being subjected to phosphorylation by cyclic AMP-dependent protein kinase. Calmodulin-dependent protein kinase II and polyanion also modulated the enzyme activity. The results suggest that tyrosine hydroxylase may be regulated similarly in both human and rat.