Muscle Cyclic AMP-dependent Protein Kinase Research Articles

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

The complete amino acid sequence of the 349-residue catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle is presented. The sequence of the subunit (Mr 40,580 including phosphate groups at threonine-196 and serine-337) was derived largely by automated Edman degradation of nine fragments generated from the carboxymethylated protein by cleavage of methionyl bonds with cyanogen bromide. These fragments were aligned along the polypeptide chain by analysis of methionine-containing tryptic peptides isolated from protein radiolabeled in vitro by [14C]methyl exchange at methionyl residues. The molecule contains only two cysteinyl residues, at positions 198 and 342. It is relatively polar, containing clusters of cationic residues toward the amino terminus and anionic residues towards the carboxyl terminus. Predictions of secondary structure suggest the presence of three major domains with approximately half of the residues occurring in alpha-helices and 12% in beta-strands.

The amino acid sequence of a hinge region in the regulatory subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase II

The amino acid sequence of a hinge region in the regulatory subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase II

Affinity labeling of catalytic subunit of bovine heart muscle cyclic AMP-dependent protein kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.

Autophosphorylation of rabbit skeletal muscle cyclic AMP-dependent protein kinase I catalytic subunit.

The catalytic subunit of rabbit skeletal muscle cyclic AMP-dependent protein kinase I can catalyze self-phosphorylation. The autophosphorylation reaction uses ATP as the phosphoryl donor, requires Mg2+, and is inhibited by polyarginine. Prior treatment of the catalytic subunit with Escherichia coli alkaline phosphatase in the presence of bovine serum albumin greatly enhances the autophosphorylation of the subunit. The protein-bound phosphate is stable in acid but labile in base. Incubation of the 32P-labeled phosphoenzyme with histones led neither to the phosphorylation of histones nor to a loss of radioactivity from the phosphoenzyme. The results suggest that the phosphoenzyme does not represent an intermediate of the phosphotransferase reaction.

Kinetic mechanism of skeletal muscle cyclic AMP-dependent protein kinase

Kinetic mechanism of skeletal muscle cyclic AMP-dependent protein kinase

Phosphorylation of casein by cyclic AMP-dependent protein kinase

The catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein transferase) has been tested on a variety of caseins. The B variant of β-casein was phosphorylated at a much greater rate than other β-caseins, α s1-caseins, and κ-caseins. Whole casein homozygous for β-casein B was phosphorylated at 2.5 times the rate of commercial whole casein. Gel electrophoresis experiments indicate that β-casein is the predominant component phosphorylated in commerical casein. It is therefore suggested that phosphorylation of whole casein depends on its content of the specific genetic variant, β-casein B.

Artifactual stimulation of cyclic AMP-dependent protein kinase activity by the heat-stable protein kinase “Inhibitor”

Phosphoryl incorporation from ATP into histone catalyzed by muscle cyclic AMP-dependent protein kinase is decreased in presence of heat-stable protein kinase inhibitor from rat liver. However, low concentrations of the inhibitor markedly stimulate the activity of this enzyme. We show here that the latter effect is an artifact of current assay procedures and discuss the important implications of this phenomenon.

Substrate specificity of the cyclic AMP-dependent protein kinase.

The protein substrate specificity of the catalytic subunit of rabbit skeletal muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) has been studied using genetic variants of beta casein. It was found that beta casein-B was phosphorylated at a much greater rate than beta caseins A1, A2, A3, or C. The enhanced phosphorylation of beta casein-B, as compared with the most common variant A2, was attributed to an arginine substitution for a serine at position 122, which caused a nearby residue, serine 124, to become a phosphorylation site for the protein kinase. These results further support the concept that the local primary structure is important in specificity and that arginine may be a specific determinant common to all the local phosphorylation site sequences recognized by the cyclic AMP-dependent protein kinase.

Effect of denaturation on the susceptibility of proteins to enzymic phosphorylation

Heat-denatured chicken egg white lysozyme and the reduced carboxymethylated maleylated derivative of this protein were found to serve as substrates for rabbit skeletal muscle cyclic AMP-dependent protein kinase. The native form of the protein was not a substrate. Two phosphoryl groups per mole of lysozyme were incorporated in the reaction. It was determined that the phosphoryl moieties were bound to serine 24 and serine 50 in the modified protein. Serine 24 was phosphorylated approximately 3 times as fast as serine 50. Reduced carboxymethylated maleylated derivatives of bovine serum albumin, phosphorylase b, and creatine kinase also served as substrates for the protein kinase whereas their native forms did not. The reduced carboxymethylated maleylated derivative of the inhibitory subunit of troponin was a poorer substrate than the native form of the protein. Maleylated histones F1 and F2b were also poorer substrates than the nonderivatized forms. The significance of these experiments with reference to the specificity of cyclic AMP-dependent protein kinase is discussed.

Dissociation of rabbit muscle cyclic AMP-dependent protein kinase into catalytic and regulatory subunits by p-chloromercuribenzoate and methylmercuric chloride

. Binding of cyclic AMP to the regulatory subunit of the inactive holoenzyme causes dissociation into free regulatory subunits and active free catalytic subunits. Preincubation of a num- ber of protein kinases with their protein substrates has also been shown to lead to their activation, prob- ably by promoting dissociation of catalytic and regu- latory subunits, [2-S]. However there is little infor- mation on the nature of the interaction between the subunits in the intact holoenzyme, despite some evidence that sulfhydryl groups may be involved [6]. In this paper it is shown that preincubation of rabbit muscle protein kinase with low concentrations of p-chloromercuribenzoate (PCMB) or methylmer- curie chloride causes dissociation of the holoenzyme into free catalytic and regulatory subunits. 2. Materials and methods Rabbit muscle protein kinase was purified as de- scribed by Walsh et al. [7] up to the DE 52 cellulose chromatography stage. When assayed with whole calf thymus histone as described below in the presence of 1O”M cyclic AMP the preparation catalysed the transfer of 2.7 nmol of phosphate to histone/min/mg of protein. The standard kinase assay contained 20 pmoles 238 Na-acetate, pH 6, 1 pmole

Phosphorylation of muscle proteins by cyclic adenosine 3′,5′-monophosphate-dependent protein kinase from muscle

Tissue-specific substrates for a cyclic AMP-dependent protein kinase from bovine tracheal smooth muscle have been partially characterized. The substrates are phosphorylated by purified smooth muscle kinase or by kinases found in crude extracts of bovine smooth, skeletal, and cardiac muscle. The substrates derived from and phosphorylated by smooth muscle protein kinase have a molecular weight of approximately 70 000, as determined by disc gel electrophoresis in the absence of sodium dodecyl sulfate and of approx. 20 000–25 000 when electrophoresed in the presence of sodium dodecyl sulfate. It appears that there are a liminated number of substrates for the smooth muscle cyclic AMP-dependent protein kinase.