Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

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.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Comparison of mode of activation of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase (protein kinase G) partially purified from silkworm pupae was selectively activated by cyclic GMP at lower concentrations. Nevertheless, the enzyme seemed to differ from adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) with respect to the mode of response to cyclic nucleotides. The catalytic activity and cyclic GMP-binding activity were not dissociated by cyclic GMP in a manner similar to that described for protein kinase A. The enzyme was not inhibited by regulatory subunit of protein kinase A nor by protein inhibitor. A sulfhydryl compound such as 2-mercaptoethanol or glutathione was essential for the activation by cyclic GMP, and an extraordinary high concentration of either Mg2+ (100 mM) or Mn2+ (25 mM) was needed for maximal stimulation by cyclic GMP. A polyamine such as spermine, spermidine, or putrescine could substitute partly for the cation. Kinetic analysis indicated that Km for ATP was decreased whereas Ka for cyclic GMP was increased significantly at high concentrations of the cation. The effect of the cation to decrease Km for ATP was not evident in the absence of a sulfhydryl compound. These characteristics of protein kinase G described above were not observed for protein kinase A which was obtained from the same organism.     

Functional specificity of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Adenosine 3':5'-monophosphate-dependent protein kinase partially purified from silkworm pupae shows identical functional activities with those of mammalian protein kinases; the insect and mammalian kinases are completely exchangeable in the phosphorylation of muscle glycogen phosphorylase kinase and glycogen synthetase resulting in the activation and inactivation of the respective enzymes. In contrast, guanosine 3':5'-monophosphate-dependent protein kinase obtained from the same organism is totally inactive in this role and phosphorylates different, mainly seryl and some threonyl, residues of acceptor proteins. Substrates of the latter kinase intimately involved in the regulation of biological processes have remained unknown.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) purified from silkworm pupae phosphorylated five major fractions of calf thymus histone, whereas guanosine 3':5'-monophosphate-dependent protein kinase (protein kinase G) purified from the same organism reacted preferentially with H1, H2A, and H2B histones. Amino acid analysis of the phosphopeptides which were obtained by proteolytic digestion revealed that both protein kinases A and G showed the abilities to phosphorylate the same serine hydroxyl groups in H1 and H2B histones. Both protein kinases reacted with Ser-38 in H1 histone. With H2B histone as substrate protein kinase A phosphorylated Ser-32 as well as Ser-36, whereas protein kinase G reacted preferentially with Ser-32 and the reaction with Ser-36 was very slow. H3 and H4 histones were practically inactive substrates for protein kinase G. Although H2A histone has not been analyzed, the evidence has raised a possibility that protein kinase G utilizes a portion of the substrate proteins for protein kinase A.     

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).     

Intrinsic activity of guanosine 3',5'-monophosphate-dependent protein kinase similar to adenosine 3',5'-monophosphate-dependent protein kinase. I. Phosphorylation of histone fractions.

Guanosine 3',5'-monophosphate (cyclic GMP)-dependent protein kinase partially purified from silkworm pupae reacts preferentially with H1, H2A, and H2B histones but not with H3 AND H4 histones. However, the latter can serve as substrates in the presence of a stimulatory modulator as described by Kuo and Kuo (J. Biol. Chem. 251, 4283-4286 (1976)). With H2B histone as substrate high Mg2+ concentrations (50-100 mM) are necessary for the maximum rate of reaction. Although effects of the modulator and Mg2+ vary significantly with the histone fractions employed, analysis on the phosphorylation of histone fractions provides evidence that cyclic GMP-dependent protein kinase possesses an intrinsic activity that is similar to that of adenosine 3',5'-monophosphate-dependent protein kinase.