Activation Of cGMP-dependent Protein Kinase Research Articles

14] cGMP-dependent protein kinase activation in intact tissues

Publisher Summary Unlike cAMP-kinase that dissociates into regulatory and catalytic subunits upon activation with cAMP, cGMP-kinase remains as a holoenzyme upon binding of cGMP. An increase in the activity of purified cGMP-kinase is observed after adding cGMP in sufficient amounts to occupy the cGMP-binding sites. In intact cells, the enzyme is probably activated in a similar manner. Ideally, when measuring the activation of cyclic nucleotide-dependent protein kinases in intact tissues, the equilibrium should not be disturbed by the procedures of tissue extraction or assaying. In the case of cAMP-kinase, the fact that the regulatory and catalytic subunits dissociate upon activation helps to preserve the activation state of the enzyme when it is diluted into homogenizing buffer and further diluted into a kinase reaction solution for assaying. In contrast, activated cGMP-kinase, under similar conditions, begins to lose activity, 4 probably because of the rapid dissociation of cGMP from the enzyme as a result of dilution. Therefore, special procedures are needed to minimize cGMP dissociation.

7] Purification and assay of cAMP, cGMP, and cyclic nucleotide analogs in cells treated with cyclic nucleotide analogs

To determine cAMP, cGMP, or cyclic nucleotide analog levels, this chapter develops a method to first separate the analog(s) from cAMP or cGMP by a simple chromatographic procedure followed by functionally directed assay of the respective cyclic nucleotide by either cAMP- or cGMP-dependent protein kinase activation. The sensitivity is much better than that of the cyclic nucleotide binding assays, and approaches that of the radioimmunoassays. Because high concentrations of cyclic nucleotide analogs are usually used for intact tissue incubations, trace cyclic nucleotide contaminants in commercial cyclic nucleotides may not be separated during purification and would thus interfere with the assay for the nucleotide of interest. It is therefore necessary to purify the analogs before use. Alternatively, sensitive and specific cAMP- and cGMP-dependent protein kinase activation assays are described in the chapter.

Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein.

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.

Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets.

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.

Diastereomers of adenosine 3',5'-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase.

cGMP-dependent protein kinase contains four cGMP-binding sites which are homologous to the four cAMP-binding sites of cAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3',5'-thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 microM and 94 microM, respectively. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 microM. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 microM) and cAMP (approx. KA 98 microM) but neither by the (PR) nor (PS) stereoisomer of cAMP[S]. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP-or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 microM and 73 microM, respectively. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 microM and 15 microM, respectively. These results show that both diastereomers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.

Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside

Methacholine (3 μM) and sodium nitroprusside (300 μM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 μM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0°C) and with an abbreviated incubation time (2.5 min). When assayed at 30°C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 μM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0°C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.

Tolerance towards nitroglycerin, induced in vivo, is correlated to a reduced cGMP response and an alteration in cGMP turnover

Rats were made tolerant towards nitroglycerin (GTN) by subcutaneous injections of 50 mg/kg GTN, 3 times daily for 3 consecutive days. The effects of a test dose of GTN on tension and on the cGMP response were studied in aortic preparations. The activities of the enzymes regulating cGMP turnover were also investigated. In noradrenaline (2.5 μM)-contracted tissue from GTN-treated animals, the relaxant response towards the test dose of GTN (44 μM) was reduced by about 75% as compared to the ethanol-treated control rats. The cGMP-elevating action of GTN was reduced by 55%, while no significant effect on the cAMP level could be detected. The cyclic GMP-phosphodiesterase (G-PDE) activity was increased from 770 pmol/min × mg prot (ethanol-pretreated rats) to 1340 pmol/min × mg prot (GTN-pretreated animals). The guanylate cyclase activity, stimulated with 1 mM nitroprusside, was determined with both MnGTP and MgGTP as substrate and found to be reduced by about 75% in aortic homogenates from the GTN-pretreated animals. A slight depression in cGMP-dependent protein kinase activity was detected in aortas from GTN-treated animals. However, this depression seemed to be due to an increased breakdown of the activator (cGMP) since the inclusion of 2.5 mM theophylline in the assay solution abolished the difference. These results strongly support the suggestion that cGMP acts as a mediator of GTN-induced vascular smooth muscle relaxation. The tolerance towards the pharmacological action of GTN after repeated exposure could well be explained by the reduced formation and increased rate of breakdown of cGMP as demonstrated in this study.

Purification and properties of a cGMP-dependent protein kinase from Ceratitis capitata pharate adults

1. 1. A general method for the purification of a cGMP-dependent protein kinase from Ceratitis capitata has been described. 2. 2. The molecular weight is similar to the cGMP-dependent protein kinase described in Bombyx mori eggs. 3. 3. This protein kinase activity requires the presence of a divalent cation. This activation follows the order Mg 2+ > Co 2+ > Mn 2+. The enzyme activity vs the Mg 2+ concentration exhibits a biphasic curve, similar to that described for the enzyme of other sources. 4. 4. The cGMP-dependent protein kinase activity is much lower than the cAMP-dependent activity. 5. 5. This protein kinase phosphorylates histone H2B better than other histones, total histones and protamine.

Increased noradrenergic metabolism in the cerebellum of the mouse mutant dystonia musculorum.

The neurological mouse mutant dystonia musculorum exhibits bizarre appendicular and truncal dystonia without known cerebellar histopathology. We evaluated striatal dopamine and cerebellar norepinephrine metabolism in this mutant and compared the results with those obtained in wild-type BALB/c and B6C3 controls. Tyrosine hydroxylase activity and dopamine metabolite levels (homovanillic acid and 3,4-dihydroxyphenylacetic acid) in the striatum of the mutant were similar to controls. Tyrosine hydroxylase activity and the steady-state level of 3-methoxy-4-hydroxyphenethyleneglycol, a metabolite of norepinephrine, in the cerebellum were 38% and 42-66%, respectively, greater in the mutant. However, the level of norepinephrine was similar (approximately 350 ng/g). Further, a Purkinje cell-specific marker, cGMP-dependent protein kinase, was unchanged in the mutant and no Purkinje cell pathology was observed with light microscopy. The lack of Purkinje cell derangement and similar levels of cerebellar norepinephrine and cGMP-dependent protein kinase activity suggest that increased norepinephrine metabolism in the cerebellum of this mutant is not a morphological response to gross target cell loss during morphogenesis. The observed changes may be a reaction to abnormal impulse traffic or altered input/output pathways to the mutant cerebellum during its development.

Cyclic nucleotide-dependent protein kinases and cAMP-binding protein from Ceratitis capitata brain

1. 1. Soluble protein kinases from Ceratitis capitata brains have been examined. 2. 2. Two cAMP-dependent (A1 and A2) and one cGMP-dependent protein kinase activities were fractionated through Biogel HTP chromatography and the ratio between both cyclic and nucleotide-dependent protein kinase activities was similar to those described for other Diptera. 3. 3. In addition to the protein kinases A1 and A2, another protein without enzyme activity binds cAMP. 4. 4. An optimal 25 mM Mg 2+ concentration was found for the protein kinase activity A2 in agreement with cAMP-dependent protein kinases from other sources. Histones H2B and H1 were the most efficient substrates for both cyclic nucleotide-dependent protein kinases. 5. 5. Using total histones as substrate, the cGMP-dependent protein kinase activity vs Mg 2+ concentration exhibited a biphasic shape with a maximal activity at about 100 mM Mg 2+.

A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. II. Kinetic studies on its phosphorylation by guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases.

Kinetic studies on the activity of purified cGMP-dependent protein kinase and catalytic subunit of cAMP-dependent protein kinase have been carried out using a protein termed G-substrate (see preceding paper) as the phosphate acceptor. Each enzyme catalyzed the phosphorylation of 2.0-2.1 mol of 32P/mol of G-substrate, with phosphorylation occurring primarily at threonine residues. When phosphorylation was carried out in the simultaneous presence of the two enzymes, the stoichiometry increased only slightly, to a value of 2.4, suggesting that both enzymes phosphorylated the same two sites. Initial rate studies on the phosphorylation of G-substrate by cGMP-dependent protein kinase yielded a Km of 0.21 microM and a Vmax of 2.2 mumol/min/mg. Similar studies with the cAMP-dependent protein kinase yielded a Km of 5.8 microM and a Vmax of 2.3 mumol/min/mg. cGMP-dependent protein kinase thus exhibited a high degree of specificity towards this substrate which was apparently based on selective substrate binding rather than catalytic efficacy. The activity of cGMP-dependent protein kinase towards G-substrate was maximal at pH 7.5-8.0 and a Mg2+ concentration of 1-3 mM. Activity declined sharply at high ionic strength (greater than 20 mM KCl).

Purification and subunit composition of guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung.

The guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung was purified to apparent homogeneity by affinity chromography using 8-2-aminoethylthio-cGMP coupled to Sepharose 4B. The kinase activity was purified approximately 6000-fold with an overall recovery of approximately 20%. The product isolated by affinity chromatography contained both cGMP-binding and cGMP-dependent histone kinase activity, indicating that the enzyme was not dissociated into regulatory and catalytic components by the immobilized cGMP derivative. The enzyme had a molecular weight of approximately 165,000 and a sedimentation coefficient of 7.8 S. The purified kinase displayed several characteristics similar to that of the partially purified enzyme including specificity for cGMP and stimulation by high concentrations of magnesium. On sodium dodecyl sulfate gels, only one major polypeptide chain was present having a molecular weight of approximately 81,000. This subunit bound 1 mol of cGMP and exhibited cGMP-dependent protein kinase activity. It is proposed that the native enzyme consists of two identical subunits (Mr=81,000), each of which binds cGMP and catalyzes protein phosphorylation.

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.

Guanosine 3':5'-cyclic monophosphate binding proteins in rat tissues.

Rat tissues were surveyed for proteins which bind cGMP. Binding activity was high in extracts of lung, cerebellum, and small intestine, but was low in those of liver, adipose tissue, and skeletal muscle. DEAE-cellulose chromatography resolved two peaks of cGMP-binding activity in most tissues. The binding protein in peak 1 was eluted in the flow-through volume and was most abundant in extracts of intestine. It had a sedimentation coefficient of 6S and was highly specific for cGMP at pH 7.0 (dissociation constant KD=0.05 muM). No cGMP-dependent histone kinase activity was found for this peak. The binding protein in peak 2 was eluted by 0.05-0.15 M NaCl and was the predominant binding substance in lung, cerebellum, and heart. It had a sedimentation coefficient of 8S and binding was also highly specific for cGMP, with a KD of 0.05 muM. This peak of binding activity was associated with cGMP-dependent protein kinase activity which could be purified approximately 200-fold by Sepharose 6B chromatography. Cyclic GMP dependency of kinase activity was observed only at low histone concentrations. The abundance of one or both the above binding proteins correlated with the known basal levels of cGMP in the tissues.

Isolation of stimulatory modulator from rat brain and its specific effect on guanosine 3′:5′-monophosphate-dependent protein kinase from cerebellum and other tissues

The stimulatory and the inhibitory activities of protein kinase modulator originally present in the crude modulator preparation obtained from rat brain was separated by Sephadex G-100 gel filteration. The isolated stimulatory modulator, as the crude modulator, augmented the activity of guanosine 3′:5′-monophosphate (cGMP)-dependent protein kinase from both the mammalian tissues (cerebellum, heart and lung) and the lobster tail muscle. The purified cGMP-dependent protein kinase was not activated by cGMP unless the stimulatory modulator, or the crude modulator, was added. The isolated inhibitory modulator, as the crude modulator, on the other hand, depressed the activity of adenosine 3′:5′-monophosphate (cAMP)-dependent protein kinase. It neither inhibited nor stimulated the activity of cGMP-dependent protein kinase.

47] Purification and characterization of cyclic GMP-dependent protein kinases

Many arthropod tissues are rich in cGMP-dependent protein kinases and provide favorable material for study of the properties and function of enzymes. In contrast, it has not yet been possible to detect cGMP-dependent protein kinase activity in most vertebrate tissues examined. A correlation of the relative tissue levels of cAMP and cGMP with the apparent relative tissue levels of cAMP-dependent and cGMP-dependent protein kinases would appear to be lacking. For instance, cecropia silkmoth larval fat body, a tissue in which only cyclic GMP-dependent protein kinase activity has been detected, has a ratio of cGMP to cAMP of about 0.4; in contrast, rat cerebellum and lung, tissues in which only cAMP-dependent protein kinase has been detected, have a cGMP to cAMP ratio of about 0.7, highest of any of the mammalian or arthropod tissues examined. This lack of correlation is not entirely surprising, when one considers the great difficulty of estimating levels of enzyme activity in vivo from studies of broken cell preparations.