Characterization of cyclic GMP-binding sites of cyclic GMP-dependent protein kinase by rapid filtration assay.

The kinetics of cyclic [3H]GMP binding to the purified cyclic GMP-dependent protein kinase (cG kinase) were studied by using the rapid filtration assay method with polyethyleneimine-treated glass filters (method A), and the data were compared with those of the (NH4)2SO4 precipitation procedure (method B), which has been used for many previous studies on cyclic GMP binding to cG kinase. Each method gave a similar stoichiometry of approx. 2 mol of cyclic GMP/mol of cG kinase subunit; however, other binding kinetics obtained with these two methods were different. The dissociation of bound cyclic [3H]GMP from the kinase showed a single slow component when method A was used, whereas rapid and slow dissociation components were observed with method B. The Scatchard plot of cyclic [3H]GMP binding with method A was linear with a Kd value of 11 +/- 2 nM, suggesting that the two intrachain binding sites have similar high affinity for cyclic GMP. Results obtained on cyclic nucleotide analogue specificity of the two intrachain cyclic GMP-binding sites were also different between these two methods. These findings suggest that cG kinase has two high-affinity cyclic GMP-binding sites per subunit in the native state, and that when (NH4)2SO4 is added, ostensibly to stop the binding reaction, one low-affinity site is created from one high-affinity site.     

Two different intrachain cAMP binding sites of cAMP-dependent protein kinases

The regulatory subunits of both isozymes of cAMP-dependent protein kinase bind 2 mol of cAMP/mol of monomer. cAMP dissociation studies indicate similar cAMP binding behavior for each isozyme. Each has two different intrachain cAMP binding components present in approximately equal amounts and the rate of cAMP dissociation is 5- to 10-fold slower from one site (Site 1) than from the other (Site 2). Equilibrium [3H]cAMP binding is inhibited by several competing cyclic nucleotides. Following equilibrium binding using saturating [3H]cAMP in the presence of competing nucleotide, the pattern of release of [3H]cAMP, monitored in the presence of an excess of nonradioactive cAMP, suggests site-specific selectivity of some of the cyclic nucleotides. As compared with cAMP, cIMP prefers Site 2 for both regulatory subunits, whereas N6, O2-dibutyryl-cAMP shows a similar preference only with isozyme II regulatory subunit. 8-Bromo-cAMP, 8-bromo-cGMP, and 8-azido-cAMP prefer Site 1 of both proteins. The results indicate that for each isozyme the two intrachain binding sites have different analogue specificities and cAMP dissociation rates. Site 1 or Site 2 of one isozyme has a similar but not identical cyclic nucleotide specificity and cAMP dissociation rate to the corresponding site of the other isozyme.     

8-(2-Carboxymethylthio)-cGMP, a site-1-selective compound for cGMP-dependent protein kinase

The cGMP analogue 8-(2-carboxymethylthio)-cGMP (CMT-cGMP) was synthesized and its binding to cGMP-dependent protein kinase (cGMP kinase) was studied. CMT-cGMP bound at 4 degrees C with an over 1400-fold higher affinity to site 1 than to site 2 of the native enzyme with apparent Kd values of 4.1 nM and 5.9 microM, respectively. The apparent selectivity for site 1 was about threefold less with the autophosphorylated enzyme and about sixfold with the catalytically active fragment of cGMP kinase. The apparent selectivity was confirmed by determination of the dissociation of [3H]cGMP from cGMP kinase in the presence of 1 microM CMT-cGMP at 4 degrees C. The apparent site 1 selectivity was 250-fold at 30 degrees C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 microM for site 1 and 2, respectively. CMT-cGMP stimulated the phosphotransferase activity of native and autophosphorylated cGMP kinase with Ka values of about 80 nM. About 60% of the total catalytic rate of cGMP kinase was obtained in the presence of 1 microM CMT-cGMP and 0.13 mM Kemptide. The apparent Km values for ATP and Kemptide were not affected. However, CMT-cGMP activated the enzyme to the same level as cGMP when 1.3 mM Kemptide was present. CMT-cGMP and cGMP inhibited cAMP-stimulated autophosphorylation of cGMP kinase with IC50 values of 0.7 microM and 2 microM, respectively. Neither compound stimulated autophosphorylation of cGMP kinase by itself. These results indicate that CMT-cGMP binds with high preference to site 1 of cGMP kinase and that occupation of site 1 may lead to expression of a partial enzyme activity.     

Interconvertible cGMP-free and cGMP-bound forms of cGMP-dependent protein kinase in mammalian tissues

Several vascular and nonvascular mammalian tissue extracts exhibited variable amounts of two peaks (peaks I and II) of cGMP-dependent protein kinase by NaCl elution of DEAE columns. When [3H]cGMP was added to the extracts before chromatography, a peak of protein-bound [3H]cGMP coeluted with peak II. [3H]cGMP was added to purified bovine lung cyclic nucleotide-free enzyme followed by chromatography on high performance liquid chromatography-DEAE. Two kinase peaks, the first of which represented mainly cGMP-free enzyme and the second of which represented cGMP-bound enzyme, eluted at the same positions as peaks I and II, respectively, of the crude extracts. The relative amount of peak II increased as a function of increasing the [3H]cGMP added before chromatography, and peak II could be converted partially to peak I by rechromatography. The holoenzyme is known to contain two slowly exchanging cGMP binding sites (sites 1) and two rapidly exchanging sites (sites 2). Some protein-bound [3H] cGMP found entirely in site 1 coeluted with peak I, although most of the enzyme in that peak was cGMP-free. When low [3H]cGMP was used for the initial incubation, relatively more of the protein-bound [3H] cGMP appeared in peak I and could represent binding of [3H]cGMP to only one of the two sites 1 of the kinase. The [3H]cGMP bound to the peak II enzyme completely filled both sites 1. Cyclic GMP binding to these sites caused the apparent conformational change which shifted the DEAE elution position of the enzyme. The peak II kinase was partially active and had a higher sensitivity to further cGMP activation of kinase than did the cGMP-free enzyme, suggesting that activation of kinase by binding of cGMP to site 2 was facilitated by prior binding at site 1. In fractions of the trailing edge of peak II, the kinase activity was virtually cGMP-independent, and both sites 1 and 2 were almost saturated with [3H]cGMP. These results suggested a further conformational change and direct increase in activity by binding of cGMP at site 2.     

Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP

Cyclic-GMP-dependent protein kinase contains two binding sites for cGMP, which have different affinities for cGMP. Autophosphorylation of the enzyme affects mainly the binding of cGMP to the 'high'-affinity site (site 1). The enzyme binds cAMP and cAMP stimulates the phosphotransferase activity of the native enzyme half-maximally at 44 microM. Autophosphorylation of the enzyme decreases the apparent Ka value to 7 microM. Autophosphorylation does not affect the catalytic rate of the enzyme if measured at a saturating concentration of ATP. Tritiated cAMP apparently binds at 4 degrees C to one site with a Kd value of 3 microM. Binding to the second site is not measurable. Autophosphorylation of the enzyme increases the affinity of the high-affinity site for cAMP sixfold (Kd 0.46 microM) and allows the detection of a second site. In accordance with these data the dissociation rate of [3H]cAMP from the high-affinity site is decreased from 4.5 min-1 to 1.2 min-1 by autophosphorylation. Experiments in which unlabeled cAMP competes with [3H] cGMP for the two binding sites confirmed these results. Recalculation of the competition curves by a computer program for two binding sites indicated that autophosphorylation decreases the Kd value for binding of cAMP to the high-affinity site from 1.9 microM to 0.17 microM. Autophosphorylation does not affect significantly the affinity for the second site. Kd values for site 2 varied from 17 microM to 40 microM. These results suggest that autophosphorylation of cGMP-dependent protein kinase increases the affinity of the enzyme for cAMP by affecting mainly the properties of binding site 1.     

The amino terminus regulates binding to and activation of cGMP-dependent protein kinase

The allosteric regulation of binding to and the activation of cGMP-dependent protein kinase (cGMP kinase) was studied under identical conditions at 30 degrees C using three forms of cGMP-kinase which differed in the amino-terminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4 +/- 0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino-terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP-binding sites, Km values for MgATP and the heptapeptide kemptide, and Vmax values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrations of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical Kd values of 0.2 microM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino-terminal segment. Cyclic GMP activates the native enzyme at Ka values which are identical with the Kd values for both binding sites. The activation of cGMP-dependent protein kinase is noncooperative but the Ka value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino-terminal domain.     

Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of a cGMP-dependent monomeric protein kinase from bovine aorta

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.     

Purification and characterization of an inactive form of cAMP-dependent protein kinase containing bound cAMP

By a new procedure, the holoenzyme of bovine heart type II cAMP-dependent protein kinase was purified to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A high performance liquid chromatography-DEAE purification step resolved two distinct peaks of protein kinase activity, which were designated Peak 1 and Peak 2 based on their order of elution. The two peaks exhibited similar Stokes radii and sedimentation coefficients. They had similar ratios of regulatory to catalytic subunits both by densitometric scanning of SDS-PAGE bands and by the ratios of equilibrium [3H]cAMP binding to maximal kinase activity. These results suggested that the holoenzyme of each peak contained two regulatory subunits and two catalytic subunits, although a subpopulation of holoenzyme lacking one catalytic subunit also appeared to be present in Peak 2. Assays of cAMP indicated that the Peak 1 holoenzyme was cAMP-free, but half of the Peak 2 holoenzyme cAMP binding sites contained cAMP. Determination of [3H]cAMP dissociation rates showed that the cAMP was equally distributed in binding Site 1 and Site 2 of Peak 2. Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP. Interconvertibility of the two holoenzyme peaks strongly suggested that the difference between the two peaks was caused by the presence of cAMP in Peak 2. Peak 2 holoenzyme, as compared to Peak 1, had enhanced binding in nonequilibrium [3H]cIMP and [3H]cAMP binding assays, as was expected due to the presence of cAMP and to the known positive cooperativity in binding of cyclic nucleotides to the kinase. The positive cooperativity in kinase activation, as indicated by the Hill coefficient, was greater for Peak 2 than Peak 1, but the cAMP concentration required for half-maximal activation (Ka) of each of the two peaks was very similar. In conclusion, Peak 2 is an inactive ternary complex of cAMP, regulatory subunit, and catalytic subunit, and Peak 1 is a cAMP-free holoenzyme. The cAMP-bound form may represent a major cellular form of the enzyme which is primed for activation.     

Characterization of a purified bovine lung cGMP-binding cGMP phosphodiesterase.

A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 microM) and cGMP binding (half-maximum approximately 0.2 microM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial alpha-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast greater than dipyridamole greater than 3-isobutyl-1-methyl-8-methoxymethylxanthine greater than 3-isobutyl-1-methylxanthine greater than cilostamide greater than theophylline greater than rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation.     

Studies on the interactions between the cyclic nucleotide-binding sites of cGMP-dependent protein kinase

The rate and equilibrium kinetics of [3H]cGMP binding to the two rapidly exchanging and two slowly exchanging sites of dimeric cGMP-dependent protein kinase from bovine lung were studied. As observed by McCune and Gill (McCune, R. W., and Gill, G. N. (1979) J. Biol. Chem. 254, 5083-5091), unlabeled cGMP retarded the dissociation of [3H]cGMP bound to the "slow" site. This effect was due to interaction of unlabeled cGMP with the "rapid" rather than the slow site. First, the potencies of unlabeled cGMP and a number of cGMP analogs correlated nearly perfectly with their affinities for the rapid site. Second, the rate of dissociation in the absence of unlabeled ligand was independent of the degree of saturation of the slow sites. Third, unlabeled ligand inhibited the rate of dissociation more (about 10-fold) than theoretically predicted (maximum 2-fold) from interaction between two similar sites in one macromolecule. A favorable free energy coupling appeared to exist between the rapid and slow sites but not between the slow sites. cGMP associated faster to the slow site than the rapid site. Mg/ATP decreased the rate of association to either site by 50% and increased about ten-fold the rate of dissociation from the slow site. The dissociation of cGMP from the slow site could be described by a single activation energy (Ea = 71 kJ X mol-1) for the whole temperature range (0-37 degrees C) tested. These data indicated that the cyclic nucleotide-binding sites of the cGMP-kinase are kinetically more homologous to those in the cAMP-dependent protein kinases than previously recognized.     

Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase

The specificity of the two intrasubunit cGMP binding sites of cGMP-dependent protein kinase was determined by measuring the ability of 46 cGMP analogs to compete with [3H]cGMP. Both sites of the enzyme exhibited high specificity for the ribose cyclic phosphate moiety, and lower specificity for the guanine moiety. Effects of modifications in the ribose cyclic phosphate moiety suggested that cGMP is bound at both sites by three hydrogen bonds at 2'-OH, 3'-O, and 5'-O. A negative charge in the cyclic phosphate is apparently required. Modifications of the pyrimidine part of guanine, particularly at C-1, generally caused selectivity for the rapidly exchanging site while modifications of the imidazole part of guanine at C-7 and C-8 caused selectivity for the slowly exchanging site. These increases in selectivity for a site were mainly due to losses in affinity of the other site. There was an apparent requirement of the intact amino group at C-2, particularly for the slowly exchanging site. Comparison of the molecular interactions of cAMP and cGMP with their specific protein kinases showed that both nucleotides are bound by similar forces in the 2', 3' and 5' region, both bases may be bound in syn conformation, but that each base moiety is bound by different molecular interaction, thus leading to the selectivity of the two enzymes. cGMP analogs which possessed strong selectivity for the rapidly exchanging site, but not those selective for the slowly exchanging site, stimulated the binding of [3H]cGMP. Only a few cGMP analogs were more potent than cGMP in stimulating protein kinase activity. The potency of cGMP analogs as stimulators of kinase activity correlated better with the mean binding affinity for both binding sites than with the affinity for either site alone. Two analogs added in combination were synergistic in kinase activation, particularly if one analog was selective for the slowly exchanging site and the other for the rapidly exchanging site. These observations are suggestive that cGMP binding at the rapidly exchanging site stimulates cGMP binding at the slowly exchanging site and that both sites are involved in the activation process.     

Characterization of specific binding sites for [3H]-staurosporine on various protein kinases

Binding of [3H]-staurosporine to different protein kinases was time-dependent, reversible and saturable. Scatchard analysis of saturation isotherms indicated one class of binding sites for [3H]-staurosporine with dissociation constants (KD) of 9.6, 2.0, 3.0 and 7.4 nM for protein kinase C, cAMP-dependent protein kinase, tyrosine protein kinase and calcium/calmodulin-dependent protein kinase respectively. [3H]-staurosporine binding was fully displaced by unlabelled staurosporine or the related compound K-252a whereas other protein kinase inhibitors (H-7, H-8 and W-7) did not compete with [3H]-staurosporine. These data confirm that sataurosporine shows no selectivity for different protein kinases and suggest the putative existence of distinct, specific binding sites for [3H]-staurosporine on these enzymes.     

Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites

Two S49 mouse lymphoma cell variants hemizygous for expression of mutant regulatory (R) subunits of type I cyclic AMP-dependent protein kinase were used to investigate functional consequences of lesions in the putative cAMP-binding sites of R subunit. Kinase activation properties of wild-type and mutant enzymes were compared using cAMP and six site-selective analogs of cAMP. Kinases from both mutant sublines were relatively resistant to cyclic nucleotide-dependent activation, but they were fully activable by at least some effectors. Relative resistances of the mutant kinases varied from about 5-fold for analogs selective for their nonmutated sites to as much as 700-fold for analogs selective for their mutated sites; resistance to cAMP was intermediate. Apparent affinities of wild-type and mutant R subunits for [3H]cAMP were not appreciably different, but competition experiments with site-selective analogs of cAMP suggested that binding of cAMP to mutant R subunits was primarily to their nonmutated sites. Analyses of cooperativity in cyclic nucleotide-dependent activation of mutant kinases, synergism between site I- and site II-selective analogs in activating the mutant enzymes, and dissociation of bound cAMP from mutant R subunits provided additional evidence that the mutations in these strains selectively inactivated single classes of cAMP-binding sites: phenomena attributable in wild-type enzyme to intrachain interactions between sites I and II were always absent or severely diminished in experiments with the mutant enzymes. These results confirm that R subunit sequences implicated in cAMP binding by homology with other cyclic nucleotide-binding proteins actually correspond to functional cAMP-binding sites. Furthermore, occupation of either cAMP-binding site I or II is apparently sufficient for activation of cAMP-dependent protein kinase. The presence of four functional cAMP-binding sites in wild-type kinase enhances the cooperativity and sensitivity of cAMP-mediated activation.     

Effect of cyclic nucleotide analogs on intrachain site I of protein kinase isozymes

The effects of numerous cAMP analogs present in the [3H]cAMP binding reaction on subsequent dissociation of [3H]cAMP from the regulatory subunit of cAMP-dependent protein kinase I and II were analyzed. Certain analogs with modification at either C-8 or C-2 showed relative selectivity for one (site 1) of two intrachain cAMP binding sites of both isozymes. Modification at C-6 caused selectivity for the second site (site 2). The combination of a site-1-directed and site-2-directed analog inhibited [3H]cAMP binding much more than did either analog alone. In general, there was a correlation between the site 1 selectivity and the ability of the analog to stimulate the binding of [3H]cIMP, which selects site 2. The site-1-directed analogs stimulated the initial rate of [3H]cIMP binding. The stimulatory effect was enhanced in the presence of a polycationic protein such as histone and was inhibited by high ionic strength. The type I and II isozymes exhibited large differences in analog specificity for this effect. For type I, of the analogs tested the most efficacious for stimulating [3H]cIMP binding were those containing a nitrogen atom attached to C-8, 8-aminobutylamino-cAMP being the most effective. Type II responded best to analogs containing a sulfur atom attached to C-8, 8-SH-cAMP being the most effective of those tested. The stimulatory effect was accentuated in the presence of MgATP when using type I, but this nucleotide had no effect when using type II. It is proposed that in intact tissues cAMP binding to site 1 of either isozyme stimulates the binding to site 2.     

Purification to homogeneity of protein kinase C from bovine brain--identity with the phorbol ester receptor

The calcium- and phospholipid-dependent kinase activity (protein kinase C) was isolated from bovine brains by a combination of DEAE-cellulose chromatography, gel filtration and hydrophobic chromatography on octyl-Sepharose and phenyl-Sepharose. The phorbol ester receptor co-purifies with the protein kinase C throughout the procedure yielding a homogeneous protein of 79 500 daltons on SDS-polyacrylamide gels. The purified kinase incorporated approximately 5000 nmol phosphate into substrate/min/mg protein at saturating concentrations of Ca2+ and phosphatidyl serine. Reciprocal plots of protein kinase activity at varying phosphatidyl serine concentrations were biphasic and yielded two apparent Ka values for phosphatidyl serine of 0.6-2 and 35-80 micrograms/ml). These apparent Ka values were reduced 2- to 3-fold by either diolein (20 micrograms/ml) or phorbol-12,13-dibutyrate (10 micrograms/ml). The protein binds [3H]phorbol-12,13-dibutyrate ( [3H]PDB) with high affinity (Ka = 15 nM) in a phosphatidyl serine-dependent manner. At saturating phosphatidyl serine concentrations 0.89 mol [3H]PDB are bound per mol protein. The identification of protein kinase C as the phorbol ester receptor is discussed with respect to the function and regulation of this protein.     

Characterization of the interchain and intrachain interactions between the binding sites of the free regulatory moiety of protein kinase I

The interaction between the four binding sites (two A sites and two B sites) of the regulatory subunit dimer of protein kinase I (RI2) was studied. The rate of association of c[3H]AMP to site B was slower when site A had already been occupied. Occupation of site A also retarded the rate of dissociation of c[3H]AMP from site B. This site A-B interaction was intrachain since it was observed also for a monomeric fragment of RI2. Thus, each monomer of RI2 must have one A site and one B site. Quantitative analysis of the rate constants for cAMP binding to variously liganded RI2 suggested little or no thermodynamic coupling between site A and B. This conclusion was supported by equilibrium binding data. Occupation of one A site retarded the dissociation of c[3H]AMP from the A site of the other subunit (interchain interaction). The rate kinetic constants as well as equilibrium binding data indicated a positively cooperative site A-A interaction. The interaction between cAMP and either site was enthalphy-driven (25 degrees C), the process being accompanied by a loss of entropy. The thermodynamic parameters did not support the occurrence of an abrupt conformational change at a certain level of ligandation of RI2. Half-maximal saturation of either site occurred at 1-2 nM cAMP (37 degrees C, pH 7.0, 0.15 M KCl). The concentration of RI2 did not detectably influence any binding parameters. Aging of RI2 produced a form with minimally, if at all, altered Mr, but which showed a more rapid release of c[3H]AMP bound to site B.     

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1

cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.     

The activation of expressed cGMP-dependent protein kinase isozymes I alpha and I beta is determined by the different amino-termini.

cDNA of bovine cGMP-dependent protein kinase (cGMP kinase) isozymes I alpha and I beta differ only in their amino-terminal domains (amino acids 1-89 and 1-104, respectively). Each recombinant isozyme (rI alpha and rI beta) was transiently expressed in COS-7 cells and its properties were compared with the cGMP kinase isozymes P-I and P-II purified from bovine trachea. The subunit of P-I, P-II, rI alpha and rI beta had a molecular mass of about 75 kDa. rI alpha and rI beta had S20,W values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P-I and rI alpha, and P-II and rI beta, were immunologically indistinguishable. P-I, P-II, rI alpha and rI beta had the same catalytic activity. However, rI alpha and rI beta were half-maximally activated at 0.1 microM and 1.3 microM cGMP, and 0.3 microM and 12 microM 8-bromoguanosine 3',5'-(cyclic)phosphate (Br8-cGMP), respectively. P-I and P-II had a similar shift in their apparent KA values. P-I and rI alpha bound 2 mol cGMP/mol subunit to high-affinity (site 1) and low-affinity (site 2) cGMP-binding sites. The exchange rates were 0.005-0.009 min-1 for site 1 and 3.7 min-1 for site 2. In contrast, P-II and rI beta bound and rI beta bound 2 mol cGMP/mol enzyme subunit at only two low-affinity binding sites (site 2) with k-1 values of 0.92 min-1 and 4.8 min-1. These results suggest that a change from the I alpha amino-terminal domain to that of I beta increases the apparent KA value for cGMP 10-fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long-term elevation of cGMP level.     

Structure and function studies of the cGMP-stimulated phosphodiesterase.

Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1-methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)