ABSTRACT
Metallothioneins (MTs) are a family of small, highly conserved, cysteine-rich metal-binding proteins that are important for zinc and copper homeostasis, protection against oxidative stress, and buffering against toxic heavy metals. Individual human MT isoforms are candidate biomarkers for heavy metal toxicity, and selected cancers and neurodegenerative diseases. The similar antigenicity of human MT-1 and MT-2 isoforms precludes development of antibody-based assays for their individual quantitation. Metal-based MT quantitation methods do not directly measure MT isoforms. A bottom-up mass spectrometry-based approach solves these problems by exploiting the unique masses and chromatographic properties of the acetylated N-terminal tryptic peptides of MT isoforms. These unusually hydrophilic 20- to 21-residue peptides contain five invariant cysteines. Strong cation exchange chromatography separates them from bulk internal tryptic peptides. Reversed-phase chromatography further separates them from more hydrophobic peptides of similar mass. Absolute quantitation is obtained by adding MT peptide standards alkylated with 15N-iodoacetamide to biological samples alkylated with 14N-iodoacetamide. Accurate quantitation is enhanced by dimethyl sulfide treatment to reverse oxidation of the N-terminal methionine. Originally optimized for measuring MT isoforms in cell lines, the method has been adapted to quantify MT isoforms in brain tissue and cerebrospinal fluid. The method can also be adapted for relative quantitation of MT isoforms between matched biological samples. It cannot be used to measure human MT-4 because of an arginine at position 4. Except for this type of limitation, the method is applicable to MT quantitation in many other species.
Subject(s)
Metallothionein/isolation & purification , Amino Acid Sequence , Brain , Cell Line , Chromatography, High Pressure Liquid/standards , Chromatography, Ion Exchange , Humans , Metallothionein/cerebrospinal fluid , Metallothionein/chemistry , Protein Isoforms/cerebrospinal fluid , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , Reference Standards , Sequence Analysis, Protein , Tandem Mass Spectrometry/standardsABSTRACT
A resonant mirror biosensor was used to study cyclic nucleotide-receptor interactions. In particular, a novel method was developed to determine inhibition constants (Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide-binding properties of the wild-type isolated B domain of the cAMP-dependent protein kinase type Ialpha regulatory subunit and its Ala-334-Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP-saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X-100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild-type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor-determined kinetic constants. Biosensor-determined relative inhibition constants (Ki' = Ki(cAMP)/Ki(analog)) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published Ki' values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The Ki' values for the wild-type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide-receptor interactions.
Subject(s)
Biosensing Techniques/methods , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/chemistry , Amino Acid Substitution , Animals , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic AMP-Dependent Protein Kinases/genetics , Drug Interactions , Humans , Kinetics , Models, Chemical , Mutation , Nucleotides, Cyclic/metabolism , Protein Binding , Protein Structure, TertiaryABSTRACT
The mechanism by which the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIalpha is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane-associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RIalpha gene product. A single RIalpha phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane-associated and cytosolic RIalpha. Because both R subunit preparations were 30-40% phosphorylated, this post-translational modification could not be responsible for the membrane compartmentation of the majority of RIalpha. Mass spectrometry also indicated that membrane-associated RIalpha had a higher extent of disulfide bond formation in the amino-terminal dimerization domain. No other structural differences between cytosolic and membrane-associated RIalpha were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane-associated and cytosolic RIalpha strongly suggests an interaction between RIalpha and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIalpha membrane association in the heart.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Serine/chemistry , Amino Acid Sequence , Animals , Cattle , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cysteine/chemistry , Mass Spectrometry , Membrane Proteins/chemistry , Molecular Sequence Data , PhosphorylationABSTRACT
The consensus substrate site for cAMP-dependent protein kinase (PKA) is Arg-Arg-Xaa-Ser(P)-Xaa and the autoinhibitory domain of the PKA type I alpha regulatory subunit (RI subunit) contains a similar sequence, Arg92-Arg-Arg-Arg-Gly-Ala-Ile-Ser-Ala-Glu. The italicized amino acids form a putative pseudosubstrate site (Ser is replaced with Ala), which together with adjacent residues could competitively inhibit substrate phosphorylation by the PKA catalytic subunit (C subunit). The present studies determine the contributions of Arg92-95, Ile98, and Glu101 to inhibitory potency. Amino-terminal truncation of RI subunit through Arg92 (delta1-92) or Arg93 (delta1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg94 (delta1-94), or point mutation of Arg95 within truncated mutants (delta1-93.R95A or delta1-92.R95A), caused a dramatic reduction in inhibitory potency. Truncation through Arg95 (delta1-95) had a greater effect than did replacement or deletion of Arg94 or Arg95 alone. Using full-length RI subunit, the inhibitory potency was reduced by replacing Ile98 with Ala, Gly, or Gln, but not by replacing it with Val. The inhibitory potency of RI subunit was unchanged when Glu101 was replaced with Ala or Gln. It is concluded that Arg94, Arg95 and, to a lesser extent, Ile98 are vital constituents of PKA autoinhibition by type I alpha R subunit.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Animals , Arginine/chemistry , Binding Sites , Cattle , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic IMP/metabolism , Isoleucine/chemistry , Mutagenesis, Site-Directed , Sequence Deletion , Structure-Activity RelationshipABSTRACT
The carboxyl-terminal 19 amino acids of the type I alpha regulatory subunit (RI alpha) of cAMP-dependent protein kinase (PKA) were investigated to determine their contributions to cAMP selectivity. The parent RI alpha subunit contained an Ala to Thr mutation at position 334 so that it would bind both cAMP and cGMP with high affinity. Stop codons were introduced into the parent cDNA construct at positions corresponding to Val-375, Asn-372, Gln-370, and Cys-360. The purified, bacterially expressed proteins were characterized for their cAMP and cGMP dissociation properties. Site-selective cAMP analogs were used to compete against [3H]cAMP binding to the mutant RI alpha subunits to correctly assign fast and slow dissociation t1/2 values to the A and B domains. A greater than 60-fold drop in B domain t1/2 in the Asn-372-stop to Gln-370-stop transition implicated Tyr-371 as an important cAMP-binding determinant. A similar drop in [3H]cGMP t1/2 for the same transition suggested that the cGMP/cAMP selectivity was not altered. To test this further, Tyr-371 was mutated to Ala, Phe, and Arg in the parent construct. The cAMP and cGMP t1/2 values were determined, as were protein kinase activation constants (Ka) for holoenzymes formed from mutant RI alpha subunits and purified catalytic subunit. The Ka data suggested that mutation of Tyr-371 enhanced B domain cAMP selectivity. Isolated B domains containing Tyr-371-Arg or Tyr-371-Phe mutations were constructed, expressed, and purified to determine their relative inhibition constants (K'I) for cGMP vs cAMP. These data showed that B domain cAMP selectivity was minimally affected by alteration of Tyr-371. Based on these results, it is concluded that aromatic stacking is not important for determining B-domain cyclic nucleotide selectivity. It is proposed that the main function of Tyr-371 is stabilization of the B-domain cAMP-binding pocket through hydrogen bonding with Glu-324.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , Cattle , Cyclic AMP/analogs & derivatives , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Hydrogen Bonding , Kinetics , Male , Models, Molecular , Molecular Sequence Data , Mutagenesis , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Recombinant Proteins/metabolism , Testis/enzymologyABSTRACT
The species-dependent compartmentation of type I cAMP-dependent protein kinase (PKA I) and its dissociated regulatory subunit (RI) was examined in the heart by biochemical and immunohistochemical means. PKA I and RI were resolved from type II cAMP-dependent protein kinase and its regulatory subunit by DEAE-Sephacel chromatography of the supernatant and Triton X-100 soluble particulate fractions of heart homogenates. The relative amounts of holoenzymes and subunits were determined by cAMP-binding, protein kinase, 8-N3-[32P]cAMP photoaffinity labeling, and Western blot assays. Rat, rabbit, and guinea pig hearts all contained PKA I to varying degrees, but only in the supernatant fractions. Significant amounts of dissociated RI were found in the supernatant fractions, and to a lesser extent the particulate fractions, of these species. In contrast, though no PKA I was detected in the supernatant or particulate fractions of pig and beef heart, half of the cAMP-binding activity in the particulate fraction was attributed to RI. The results suggest that RI may associate with membrane fractions when it is not associated with the PKA catalytic subunit. Immunohistochemical studies of tissue sections from pig, beef, and rat cardiac ventricle using antibodies directed against RI also revealed species-dependent localization of RI. Cardiac myocyte intercalated discs were stained in pig and beef sections with additional sarcolemmal staining in beef sections. Rat ventricle, which contained large amounts of supernatant PKA I, showed nuclear staining. The localization of RI to cardiac myocyte intercalated discs and sarcolemma in certain species suggests a role(s) for this subunit in mediating cAMP-regulated events in these regions.
Subject(s)
Carrier Proteins/analysis , Cyclic AMP-Dependent Protein Kinases/analysis , Intracellular Signaling Peptides and Proteins , Myocardium/enzymology , Animals , Autoradiography , Azides/metabolism , Blotting, Western , Carrier Proteins/isolation & purification , Cattle , Cell Compartmentation , Cell Fractionation , Chromatography, Ion Exchange , Cyclic AMP/analogs & derivatives , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase Type II , Cyclic AMP-Dependent Protein Kinases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Guinea Pigs , Heart Ventricles , Immunohistochemistry , Myocardium/cytology , Photoaffinity Labels , Protein Binding , Rabbits , Rats , Rats, Sprague-Dawley , Sarcolemma/enzymology , SwineABSTRACT
Cardiac sarcolemmal vesicles purified from bovine and porcine left ventricles contained approximately 45 pmol of cAMP-dependent protein kinase (PKA) regulatory (R) subunit per milligram membrane protein based on [3H]cAMP-binding activity. Less than 26% of this activity was complexed with the catalytic subunit forming the type H holoenzyme of PKA. The remainder was contributed by the free type I R subunit (RI). Purification of sarcolemma with buffers containing 0.15 M NaCl instead of 0.75 M NaCl did not affect the ratio of RI to RII, nor did it increase the total amount of membrane-associated cAMP-binding or kinase activity. Canine, rabbit, and rat heart sarcolemma also contained RI, but in highly varying proportions compared with RII as determined by 8-N3-[32P]cAMP photoaffinity labeling. Analysis of sarcolemmal vesicles from isolated porcine ventricular myocytes demonstrated that this cell type was the source of the membrane-associated RI. The results indicate that sarcolemmal RI must be considered as a factor that could influence the varied responses of the heart to agents that elevate intracellular cAMP.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/analysis , Myocardium/enzymology , Sarcolemma/enzymology , Adenosine Triphosphatases/analysis , Affinity Labels , Animals , Calcium-Transporting ATPases/analysis , Cattle , Chromatography, Ion Exchange , Cyclic AMP-Dependent Protein Kinases/chemistry , Dogs , Heart Ventricles , Rabbits , Rats , Sodium-Potassium-Exchanging ATPase/analysis , Species Specificity , SwineABSTRACT
We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.
Subject(s)
Acyl-CoA Dehydrogenases/metabolism , Acyl Coenzyme A/metabolism , Acyl Coenzyme A/pharmacology , Acyl-CoA Dehydrogenase , Acyl-CoA Dehydrogenases/antagonists & inhibitors , Acyl-CoA Dehydrogenases/chemistry , Acyl-CoA Dehydrogenases/isolation & purification , Animals , Databases, Factual , Fatty Acids/chemistry , Fatty Acids/metabolism , Flavin-Adenine Dinucleotide/metabolism , Humans , Kidney/enzymology , Kinetics , Liver/enzymology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrophotometry , SwineABSTRACT
A 14.4-kDa cAMP-binding fragment was generated during bacterial expression and purification of recombinant bovine cAMP-dependent protein kinase type I alpha regulatory subunit (RI alpha). The full-length RI alpha from which the fragment was derived contained a point mutation allowing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH2 terminus of the fragment was Ser-252, indicating that it encompassed the entire predicted B domain. Although the [3H]cAMP and [3H]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact RI alpha, the [3H]cAMP exchange rate was comparable to that of the B domain of full-length RI alpha containing an unoccupied A domain. A plasmid encoding only the isolated B domain was overexpressed in Escherichia coli, and a monomeric form of the B domain was purified that had identical properties to the proteolytically generated fragment, indicating that all of the elements for the high-affinity cAMP-binding B domain are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could be reverted to the monomer by incubation in 2-mercaptoethanol. Dimerization caused an approximate fivefold increase in the rate of cyclic nucleotide exchange relative to the monomer. The results show that an isolated cAMP-binding domain can function independently of any other domain structures of the R subunit.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cattle , Chromatography, Ion Exchange , Cloning, Molecular , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic GMP/metabolism , DNA Primers , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Kinetics , Molecular Sequence Data , Molecular Weight , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Point Mutation , Polymerase Chain Reaction , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
cAMP- and cGMP-dependent protein kinases are homologous proteins and are predicted to exhibit very similar three-dimensional structures. Their cyclic nucleotide binding domains share a high degree of amino acid sequence identity. cAMP- and cGMP-dependent protein kinases are activated relatively specifically by cAMP and cGMP, respectively; and a single alanine-threonine difference between cAMP- and cGMP-binding domains partially accounts for this specificity. Thus, it would be expected that cAMP and cGMP mediate separate physiological effects. However, owing in part to the lack of absolute specificity of either enzyme and to the relatively high level of cAMP or cGMP in certain tissues, it is also possible that either cyclic nucleotide could cross-activate the other kinase. Increases in either cAMP or cGMP cause pig coronary artery relaxation. However, only cGMP-dependent protein kinase specific cyclic nucleotide analogues are very effective in causing relaxation, and cAMP elevation in arteries treated with isoproterenol or forskolin activates cGMP-dependent protein kinase, in addition to cAMP-dependent protein kinase. Conversely, increases in either cAMP or cGMP cause Cl- secretion in T-84 colon carcinoma cells, and the cGMP level in T-84 cells can be elevated sufficiently by bacterial enterotoxin to activate cAMP-dependent protein kinase. These results imply specific regulation of cAMP- and cGMP-dependent protein kinases by the respective cyclic nucleotides, but either cyclic nucleotide is able to cross-activate the other kinase in certain tissues.
Subject(s)
Cyclic AMP/pharmacology , Cyclic GMP/pharmacology , Protein Kinases/metabolism , Animals , Binding Sites , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Enzyme Activation , Protein Kinases/chemistryABSTRACT
Smooth muscle preparations of human aorta or pig coronary arteries contain nearly equal amounts of cGMP-dependent protein kinase isozymes (cGMP kinase I alpha and I beta). In order to understand the roles of these isozymes in relaxing vascular smooth muscle, several new cGMP analogs were synthesized and tested for potencies in activating each enzyme and in relaxing pig coronary arteries. Analogs modified with a derivatized phenylthio group at the 8-position were as much as 72-fold more potent in activating purified cGMP kinase I alpha than cGMP kinase I beta. Electron-donating substituents, such as hydroxy, amino, and methoxy, on the phenyl ring enhanced the potencies of these analogs in activating cGMP kinase I alpha. The most potent of these cGMP analogs [8-(4-hydroxyphenylthio)-cGMP] was 17 times more potent (EC50 = 1.1 microM) as a muscle relaxant than the most efficacious analog tested previously. Among derivatives with an 8-halo group, 8-iodo-cGMP was the most potent compound (Ka = 9 nM for I alpha and 122 nM for I beta) for both I alpha and I beta. Analogs modified at the 1,N2-position or at both the 1,N2-and 8-positions of cGMP were highly potent for activating both isozymes. Within this group, 8-I-beta-phenyl-1,N2-etheno-cGMP had Ka values of 22 nM and 17 nM for cGMP kinase I alpha and I beta, respectively, whereas the Ka values of cGMP were 110 nM and 250 nM for the two isozymes. 8-I-beta-phenyl-1,N2-etheno-cGMP was the most potent muscle relaxant tested, with EC50 of 0.4 microM. For all cGMP analogs tested, there was a positive correlation between potency for activation of cGMP kinase I alpha and that for relaxation of pig coronary arteries. Assuming that the kinase assay conditions yielded a cyclic nucleotide specificity similar to that which would exist in intact cells, it was concluded that the cGMP kinase I alpha isozyme mediates the relaxation of pig coronary artery smooth muscle caused by cGMP elevation. However, an additional role for cGMP kinase I beta in the relaxation process could not be ruled out.
Subject(s)
Coronary Vessels/physiology , Cyclic GMP/analogs & derivatives , Isoenzymes/metabolism , Muscle, Smooth, Vascular/physiology , Protein Kinases/metabolism , Animals , Arteries/drug effects , Arteries/enzymology , Arteries/physiology , Chromatography, Liquid , Coronary Vessels/drug effects , Coronary Vessels/enzymology , Enzyme Activation , Humans , In Vitro Techniques , Muscle Relaxation/drug effects , Muscle Relaxation/physiology , Muscle, Smooth, Vascular/drug effects , SwineSubject(s)
Cyclic AMP/metabolism , Cyclic GMP/metabolism , Protein Kinases/metabolism , Proteins/genetics , Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Biological Evolution , Molecular Sequence Data , Protein Conformation , Receptors, Cyclic AMP/metabolism , Sequence Homology, Nucleic AcidABSTRACT
The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.
Subject(s)
Cyclic AMP/metabolism , Cyclic GMP/metabolism , Mutation , Protein Kinases/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites/genetics , Cattle , Enzyme Activation , Molecular Sequence Data , Protein Kinases/metabolism , Sequence Alignment , Sequence Homology, Nucleic Acid , Substrate Specificity/geneticsABSTRACT
Discrimination between cAMP and cGMP is a critical feature of cAMP- and cGMP-dependent protein kinases. An alanine/threonine difference in the cyclic nucleotide-binding sites has been proposed to provide a structural basis for this functional distinction. Site-directed mutagenesis of this alanine to a threonine in a cAMP-binding site of cAMP kinase produced a mutant with markedly increased cGMP affinity as determined by cGMP binding and protein kinase activation assays. Studies of other mutants at this position support the role of the threonine hydroxyl group as the component that enhances cGMP binding affinity.
Subject(s)
Cyclic GMP/metabolism , Mutation , Protein Kinases/genetics , Alanine , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cattle , Cyclic AMP/metabolism , Kinetics , Macromolecular Substances , Male , Molecular Sequence Data , Oligonucleotide Probes , Protein Kinases/metabolism , Testis/enzymology , ThreonineSubject(s)
3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Carrier Proteins/metabolism , Cyclic GMP/metabolism , Eye Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Ion Channels , Protein Kinases/metabolism , Second Messenger Systems , Animals , Biological Evolution , Cattle , Cyclic Nucleotide-Gated Cation Channels , Isoenzymes/metabolism , Lung/enzymology , Organ Specificity , Phosphorylation , Photoreceptor Cells/metabolism , Protein Conformation , Protein Kinases/genetics , Protein Processing, Post-Translational , RatsABSTRACT
Mammalian cGMP- and cAMP-dependent protein kinase show considerable similarity in amino acid sequence, although they specifically bind different cyclic nucleotides. Results of cGMP analogue binding experiments, combined with modeling of the cGMP binding sites by analogy to the structure of the homologous catabolite gene activator protein, suggest that a threonine residue forms a hydrogen bond with the 2-NH2 of cGMP. This threonine is invariant in all cGMP binding domains, but the corresponding residue in 23 out of 24 cAMP binding sites of protein kinases is alanine, which cannot form the same hydrogen bond. This alanine/threonine difference has the potential for discriminating between cAMP and cGMP and may be important in the evolutionary divergence of cyclic nucleotide binding sites.
Subject(s)
Cyclic AMP/metabolism , Cyclic GMP/metabolism , Protein Kinases/metabolism , Alanine , Amino Acid Sequence , Animals , Binding Sites , Biological Evolution , Humans , Models, Chemical , Molecular Sequence Data , Molecular Structure , Sequence Homology, Nucleic Acid , ThreonineABSTRACT
Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.
Subject(s)
Cyclic AMP/pharmacology , Genes , Phosphoenolpyruvate Carboxykinase (GTP)/genetics , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Animals , Base Sequence , Cell Line , Chromosome Deletion , Cloning, Molecular , Molecular Sequence Data , Mutation , Promoter Regions, Genetic/drug effects , Regulatory Sequences, Nucleic Acid/drug effects , TransfectionABSTRACT
The cAMP-containing phosphoform of the regulatory subunit (RII) of type II cAMP-dependent protein kinase from rat liver has been reported to have intrinsic DNA topoisomerase I activity. We found that highly purified RII preparations from eight different sources, including rat liver, contained no detectable topoisomerase I activity. Topoisomerase I exhibited an overlapping peak of activity with RII when rat liver extracts were fractionated by diethylaminoethyl-cellulose chromatography. Topoisomerase I activity was separated from RII by subsequent cAMP affinity chromatography. The results indicate that the regulatory subunit of cAMP-dependent protein kinase does not contain intrinsic topoisomerase I activity.
Subject(s)
Cyclic AMP/pharmacology , DNA Topoisomerases, Type I/isolation & purification , Liver/enzymology , Protein Kinases/isolation & purification , Adenosine Triphosphate/pharmacology , Animals , Bacteriophage phi X 174/genetics , Cattle , Chromatography, DEAE-Cellulose , DNA, Superhelical/metabolism , DNA, Viral/metabolism , Electrophoresis, Polyacrylamide Gel , Haplorhini , Lung/enzymology , Male , Myocardium/enzymology , Phosphorylation , Rabbits , Rats , Rats, Inbred Strains , SwineABSTRACT
A novel protein kinase which specifically binds single strand DNA was identified in rat liver by chromatography on double strand- and single strand- DNA cellulose. This protein kinase activity was stimulated by cAMP and was inhibited by the heat stable inhibitor, suggesting it was a cAMP-dependent protein kinase. Isoelectric focusing studies confirmed that the single strand DNA-binding protein kinase was indeed a cAMP-dependent protein kinase and had the same pI as cAMP-dependent protein kinase, Type II. The DNA binding capacity of this kinase was primarily localized in the regulatory subunit. These results support the recent hypothesis that in addition to regulating enzymatic activity by phosphorylating proteins, cAMP-dependent protein kinase, Type II, may regulate mammalian gene expression through a mechanism similar to that in prokaryotes.