Comparison of cyclic nucleotide binding to adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate-dependent protein kinases.

Binding sites for [3H]cAMP on purified regulatory dimers of type II A-kinase (II-R2) are independent as assessed by equilibrium binding (KD = 6 +/- 1.3 nM at pH 7.2, 25 degrees; nH = 1.0) and by the lack of effect of unlabeled cAMP on dissociation rate (kd = 1.0 X 10(-3) sec -1 at pH 7.2, 25 degrees). In contrast, binding sites for [3H]cGMP on purified G-kinase displayed positively cooperative interactions in both equilibrium and dissociation assays with convex upward Scatchard plots, an nH of 1.6 and a dissociation rate (kd = 6.2 X 10(-3) sec-1 at pH 6.8, 0 degree) which was slowed by excess unlabeled cGMP (kd = 1.13 X 10(-3) sec-1 at pH 6.8, degree). Calculated transition state free energies of dissociation revealed that dissociation of nucleotide from G-kinase in the presence of cGMP was restrained by an energy barrier (20.8 kcal.mol-1) similar to that of II-R2 (20.9 kcal.mol-1), whereas dissociation from G-kinase without excess nucleotide occurred more easily (18.9 kcal.mol-1).     

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.     

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.     

Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.

In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.     

Interaction of cyclic adenosine 3':5'-monophosphate with protein kinase. Equilibrium binding models.

A number of potential models for the interaction of cyclic AMP with protein kinase (RC or R2C2) have been examined. These include: Model 1, the simultaneous binding of cyclic AMP and release of C (catalytic subunit) from an independent RC protomer; Model 2, dissociation of an independent RC protomer prior to cyclic AMP binding to R (regulatory subunit); Model 3, cyclic AMP binding to RC prior to the dissociation of C; Model 4, random binding of cyclic AMP and dissociation of C with an interaction factor alpha less than 1; Model 5, release of 2C concomitant with the binding of one cyclic AMP to R2C2 followed by binding of the second cyclic AMP to the vacant R subunit; and Model 6, the simultaneous binding of cyclic AMP and release of C from one RC protomer resulting in a greater "affinity" of the other RC protomer for cyclic AMP, i.e., a cooperative version of Model 1. All the above models yield [cyclic AMP]0.5 values that increase with increasing protein concentration and Hill plots with average slopes equal to or less than 1.0 in the usual experimental range (10 to 90% of saturation). The Hill plots can be nonlinear, but for each model the exact shape of the plot changes in a characteristic (diagnostic) manner with changing protein concentration. Skeletal muscle protein kinase yields relatively linear Hill plots with napp values greater than 1.0. Consequently, Models 1 to 6 are not likely candidates. However, Model 2 is an excellent alternative model for proteins that display "negative cooperativity" with respect to the binding of a ligand. The properties of several "linear", "tetrahedral", and "all-or-nothing" cooperative models have also been examined. These include Models 7, A, B, and C and 8, A, B, and C which are cooperative versions of Models 2 and 3, respectively, and Model 9, a cooperative version of random Model 4. Model 9 is the most general model from which all others can be derived. Models 9 and 7, A, B, and C in which the prior dissociation of C greatly enhances or is an absolute requirement for cyclic AMP binding to R, are likely candidates for skeletal muscle protein kinase. All four of these models are capable of yielding Hill plots with average slopes greater than 1, and napp values that decrease with increasing protein concentration (in agreement with published data). In addition, in all four models the tight binding of MgATP to R2C2 yields decreased napp values and increased [cyclic AMP]0.5 values (also consistent with published data).     

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.     

Binding of adenosine 3',5'-monophosphate dependent protein kinase regulatory subunit to immobilized cyclic nucleotide derivatives.

Several cyclic nucleotide derivatives with aminoalkyl side chains attached to the purine ring were synthesized and their interactions with adenosine 3',5'-monophosphate (cAMP) dependent protein kinase were studied before and after immobilization to CNBr-activated Sepharose 4B. The soluble N6-substituted derivatives were as effective as cAMP itself in activating protein kinase and were more effective than 8-substituted cAMP derivatives, whereas the 2-substituted cAMP derivatives and the cGMP derivatives were the least effective. All of the synthetic derivatives tested were poor substrates for beef heart phosphodiesterase being hydrolyzed at rates less than 2% for that of cAMP itself. Utilizing methodology developed to evaluate the affinity of protein kinase for immogilized cyclic nucleotides it was found that all of the immobilized cyclic nucleotides interacted with protein kinase in a biospecific manner as judged by the following criteria: (1) the immobilized cyclic nucleotides competed with cAMP for the binding sites on protein kinase; (2) the analogous spacer-arm did not compete; and (3) the effects of enzyme concentration, MgATP, and cleavage of the cyclic phosphate ring on the interactions of protein kinase with the immobilized cyclic nucleotides were the same as previously shown for free cAMP. In addition, the immobilized ligands were bound with the same order of effectiveness as the analogous soluble ligand. The observed Ka for the activation of 0.005 muM protein kinase by N6-H2N(CH2)2-cAMP was increased from 0.23 to 3 muM by the process of immobilization. This increase was unaffected by the coupling density and spacer-arm length. The observed Kb for 0.10 muM protein kinase binding to immobilized N6-H2N(CH2)2-cAMP was increased as the molecular sieving exclusion limit of the matrix used was decreased indicating that at least part of this decrease in apparent affinity upon immobilization is due to exclusion of the enzyme from a portion of the matrix and therefore of the immobilized ligand molecules.     

DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.     

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.     

Reversibility of adenosine 3':5'-monophosphate-dependent protein kinase reactions.

Using a homogeneous enzyme from rabbit skeletal muscle, it has been demonstrated that the cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase reaction is reversible. In addition to the phosphorylated protein substrate, the reverse reaction requires Mg2+, ADP, and cyclic AMP when the holoenzyme is used as the source of enzyme. It is independent of cyclic AMP when the catalytic subunit of the protein kinase is used. The optimum pH for the reverse reaction with 32P-labeled casein as the substrate is 5.7, essentially the same as that for the forward reaction. Among the nucleotide subtrates tested, ADP serves as the best phosphoryl group acceptor. The Km of the enzyme for ADP is 3.3 mM and that for 32P-casein is 1.7 mg/ml. The equilibrium constant at 30 degrees is approximately 0.042 at a magnesium concentration of 10 mM and a pH of 6.9. This result indicates that the free energy of hydrolysis (deltaG0obs) of the phosphorylated protein substrate is relatively high, i.e. approximately -6.5 kcal/mol under these conditions.     

Phosphorylation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase regulates myosin light chain kinase

Smooth muscle myosin light chain kinase, purified to homogeneity, has a molecular weight of 130,000 +/- 5,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme has a specific activity under maximal conditions of 30 mumol Pi transferred to myosin light chain/mg kinase/min at 24 C and is totally dependent on calmodulin and calcium for activity. Incubation of myosin kinase with the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase results in the covalent incorporation of up to one mol of phosphate per mol of myosin kinase in the absence of bound calmodulin. Limited tryptic digestion of the radioactively labeled kinase indicates that all of the label has been incorporated into a single tryptic peptide (mol wt approximately 22,000), suggesting that a single site is being phosphorylated. Phosphorylation of myosin kinase lowers the rate at which the kinase phosphorylates myosin light chain. The lower rate of light chain phosphorylation is due to a weaker binding of calmodulin to the phosphorylated kinase than to the unphosphorylated kinase. Cyclic adenosine 3':5'-monophosphate-dependent phosphorylation of the kinase actin-myosin interaction represents a possible link between hormonal binding to smooth muscle receptors and muscle relaxation. A scheme for this sequence of events is presented.     

Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase.

The catalytic subunit of rabbit skeletal muscle cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been isolated in pure form. It has a molecular weight of 41,300, as determined by sedimentation equilibrium, which is in good agreement with the value of 41,000 determined by electrophoresis in the presence of sodium dodecyl sulfate. Sedimentation velocity determinations indicate that the subunit has an S20,w value of 3.12 which is essentially independent of protein concentration. These experiments are interpreted as indicating that the catalytic subunit dissociated from the holoenzyme exists as a monomer in solution. The least abundant amino acid is half-cystine, which was calculated to be present at 2.8 mol/mol of protein. The sulfhydryl reagents, N-ethylmaleimide, p-chloromercuribenzoic acid, and 5,5'-dithiobis(2-nitrobenzoic acid) inhibit the enzymatic activity of the subunit; inhibition by the two latter compounds can be reversed by 2-mercaptoethanol. Binding of 1 mol of N-ethylmaleimide/mol of protein results in almost complete inhibition. The isolated catalytic subunit contains 2.2 mol of tightly bound phosphate/mol of protein. Identification of either O-phosphoserine or O-phosphothreonine after partial acid hydrolysis indicates that at least part of the endogeneous phosphate exists as the phospho ester of one of these amino acids. Two peaks of catalytic activity corresponding to isoelectric points of pH 7.4 and 8.5 were identified by isoelectric focusing. Both forms utilize the same substrates and have similar sedimentation constants.