Characterization and regulation of heart adenosine 3':5'-monophosphate-dependent protein kinase isozymes.

There is broad species variation in the type of cAMP-dependent protein kinase isozyme present in supernatant fractions of heart homogenates as determined by DEAE-cellulose chromatography, Isozyme I, which elutes at less than 0.1 M NaCl, is predominant in mouse and rat hearts; while isozyme II, which elutes at greater than 0.1 M NaCl, is the predominant type in beef and guinea pig. Human and rabbit hearts contain about equal amounts of the two types. The type I heart kinases are more easily dissociated into free regulatory and catalytic subunits by incubation with histone than are the type II kinases, and the separated regulatory and catalytic subunits of isozyme II of rat heart reassociate more rapidly than the subunits of isozyme I under the conditions used. The data from several experiments using rat heart indicate that the basal activity ratio of the protein kinase in crude extracts (approximately 0.15) is due mainly to basal endogenous cAMP and that cAMP elevation accounts entirely for the epinephrine effect on the enzyme. Addition of epinephrine and 1-methyl-3-isobutylxanthine to the perfusate causes a rapid (1 min) increase in cAMP, active supernatant protein kinase, and active phosphorylase in perfused hearts of both rat (mainly isozyme I) and guinea pig (mainly isozyme II). The elevation percentage in cAMP is about the same in the two species, but the increase in active protein kinase is greater in rat heart. If hearts from either animal are perfused continually (10 min) with epinephrine (0.8 muM) and 1-methyl-3-isobutylxanthine (10 muM), the cAMP level, active protein kinase, and active phosphorylase remain elevated. Likewise, all parameters return rapidly to the basal levels when epinephrine and 1-methyl-3-isobutylxanthin are removed. Most of the epinephrine effect on the rat heart supernatant kinase is retained at 0 degrees if cAMP is removed by Sephadex G-25 chromatography, although this procedure completely reverses the epinephrine effect in the guinea pig heart. The epinephrine effect on the rabbit heart kinase (approximately equal amounts of isozymes I and II) is partially reversed by Sephadex G-25. These species differences can be accounted for by differences in association-dissociation behavior of the isozymes in vitro. The data suggest that epinephrine causes activation of both isozymes. The activity present in the particulate fraction comprises nearly half of the total cAMP-dependent protein kinase activity in homogenates of rabbit heart. Triton X-100 extracts of low speed particulate fractions from hearts of each species tested, including rat heart, contain predominantly or entirely the type II isozyme, suggesting differences in intracellular distribution of the isozymes. The binding of the protein kinase to the particulate fraction is apparently due to the properties of the regulatory subunit component. Differences in topographical distribution of the isozymes could provide for differences in either physiological regulation or substrate specificity.     

Properties of epinephrine-induced activation of cardiac adenosine 3',5'-monophosphate-dependent protein kinase.

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (-cyclic AMP/"cyclic AMP) of 12 000 X g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 microgram/kg) resulted from an increase in independent protein kinase activity (-cyclic AMP) without a change in total protein kinase activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 microgram/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.     

Regulation of adenosine 3:5-monophosphate-dependent protein kinase.

The effects of epinephrine, glucagon, insulin and 1-methyl-3-isobutylxanthine on adenosine 3:5-monophosphate (cAMP)-dependent protein kinase activity were investigated in the perfused rat heart. The conditions for homogenization of heart tissue and assay of protein kinase are described. The activation state of the enzyme is expressed as the ratio of the rate of phosphorylation of histone in the absence to that in the presence of 2 mu-M cAMP. This activity ratio is stable in crude homogenates over 15 min of incubation; it is not affected by up to 30-fold dilution of the tissue volume. The ratio is elevated to a variable degree in hearts taken immediately from the animal but falls to a stable, basal level of 0.15 to 0.20 after 15 min of perfusion in vitro. An optimal concentration of epinephrine (10 mu-M) in the perfusate elevates cAMP from 0.5 to 1.3 nmol per g of tissue and increases the protein kinase activity ratio from 0.20 to 0.65. When hearts are perfused with a steady, submaximal concentration of epinephrine (0.4 mu-M), the level of cAMP and the protein kinase activity ratio rise in parallel within 15 s and remain elevated for at least 10 min. When epinephrine is removed from the perfusion medium, the level of cAMP and enzyme activity ratio decline rapidly to basal levels. Both glucagon and the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine also increase the cardiac cAMP levels and protein kinase activity ratio in a dose-dependent manner. Glucagon acts as rapidly as does epinephrine whereas 1-methyl-3-isobutylxanthine requires at least 30 s before any effect can be observed. Insulin by itself does not significantly affect the cyclic nucleotide level or enzyme activity. The hormone has not been observed to lower the cAMP level or protein kinase activity in the heart under any conditions tested. In concentrations of 10 microunits per ml or greater, it does, however, cause a slight rise in the tissue level of cAMP and the protein kinase activity when these have been elevated to intermediate levels by exposure to epinephrine. This effect could only be observed when hearts were treated with catecholamine and could not be detected with glucagon or 1-methyl-3-isobutylxanthine. In all cases tested, slight increases in the protein kinase activity ratio (from 0.2 to 0.3) were accompanied by much greater increases in the amount of phosphorylase in the a form (20% to 70%). It was observed that at perfusion times greater than 3 min, there was a significant reduction in phosphorylase activity even though both the cAMP level and protein kinase activity remained elevated. In these studies, changes in the protein kinase activity correlate well with the tissue cAMP levels under all conditions tested.     

Studies on functional domains of the regulatory subunit of bovine heart adenosine 3':5'-monophosphate-dependent protein kinase

The functional domains of the regulatory subunit of isozyme II of cAMP-dependent protein kinase were studied. It was shown using Edman degradation that the regulatory subunit contained a phosphorylated residue which was very close in primary sequence to the site most sensitive to hydrolysis by low trypsin concentrations as postulated previously (Corbin, J.D., Sugden, P.H., West, L., Flockhart, D.A., Lincoln, T.M., and McCarthy, D. (1978) J. Biol. Chem. 253, 3997-4003). Catalytic subunit incorporated 0.9 mol of 32P from [gamma-32P]ATP into a preparation of regulatory subunit that contained 1.1 mol of endogenous phosphate. After phosphorylation by the catalytic subunit, the regulatory subunit contained 2.2 mol of chemical phosphate. The effects of heat denaturation upon the rate and extent of phosphorylation of the regulatory subunit were compared with the effects of these treatments upon the cAMP binding and inhibitory domains. These data suggested that the regulatory subunit required factors in addition to an intact phosphorylatable primary sequence in order for inhibitory activity to be expressed. Such factors might be part of the secondary or tertiary structure of the protein. These studies are discussed with respect to the mechanism of inhibition of catalytic activity, and a model of the regulatory subunit structure is proposed.     

Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase

Fluorescence intensity and anisotropy measurements using the fluorescent adenosine cyclic 3',5'-phosphate (cAMP) analogue 1,N6-ethenoadenosine cyclic 3',5'-phosphate (epsilon-cAMP) are sensitive to the dissociation of epsilon-cAMP which occurs when either the type I or the type II regulatory subunit (RI or RII) of cAMP-dependent protein kinase associates with the catalytic subunit. Studies using epsilon-cAMP show that MgATP has opposite effects on the reconstitution of both types of protein kinase: MgATP strongly stabilizes the type I holoenzyme while it slightly destabilizes the type II holoenzyme. The synthetic substrate Kemptide has a small inhibitory effect on the reconstitution of both holoenzymes when tested at 10 microM concentration. The protein kinase inhibitor has a larger effect which is especially pronounced in the reassociation of the type I enzyme. The diminished relative ability of the type I regulatory subunit to compete with the protein kinase inhibitor suggests that the combined effects of the two opposing equilibria (epsilon-cAMP and catalytic subunit binding) are different for the two types of regulatory subunits. Displacement experiments show that cAMP and epsilon-cAMP bind about equally well to the type I subunit. Slow conformational changes accompanying the binding of epsilon-cAMP by both regulatory subunits are greatly accelerated with the holoenzymes, suggesting that dissociation of the holoenzymes occurs via ternary complexes. The time courses of epsilon-cAMP binding also show the heterogeneity of binding characteristics of RII. The 37 000-dalton fragment of type II subunit retains the epsilon-cAMP binding properties of the native subunit. However, only a fraction of the fragment preparation (approximately 32% estimated from sedimentation measurements) binds the catalytic subunit well, suggesting heterogeneity of cleavage.     

Antiserum against the catalytic subunit of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases.

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.     

Bovine brain adenosine 3',5'-monophosphate dependent protein kinase. Mechanism of regulatory subunit inhibition of the catalytic subunit.

Adenosine 3',5'-monophosphate (cAMP) dependent protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins according to the following chemical equation: ATP + protein leads to phosphoprotein + ADP. The DEAE-cellulose peak II holoenzyme from bovine brain, which is composed of regulatory and catalytic subunits, is resistant to ethoxyformic anhydride inactivation. After adding cAMP, the protein kinase becomes susceptible to ethoxyformic anhydride inhibition. Ethoxyformic anhydride (2mM) inhibits the enzyme 50% (5 min, pH 6.5, 30 degrees) in the presence of 10 muM cAMP, but less than 5% in its absence. The substrate, Mg2+-ATP, protects against inactivation suggesting that inhibition is associated with modification of the active site. Addition of regulatory subunit or Mg2+-ATP to the isolated catalytic subunit also prevents ethoxyformic anhydride inactivation. These results suggest that the regulatory subunit shields the active site of the catalytic subunit thereby inhibiting it. In contrast to the bovine brain or muscle DEAE-cellulose peak II holoenzyme, the bovine muscle peak I holoenzyme is susceptible to ethoxyformic anhydride inactivation in the absence of cAMP.     

Properties of epinephrine-induced activation of cardiac adenosine 3′,5′-monophosphate-dependent protein kinase

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (—cyclic AMP/+cyclic AMP) of 12 000 × g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 μg/kg) resulted from an increase in independent protein kinase activity (—cyclic 2 AMP) without a change in total protein observ activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 μg/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.     

Activity and subcellular distribution of protein kinase dependent on adenosine 3':5'-monophosphate in liver from normal and adrenalectomized rats.

We have examined whether glucocorticoids control the activity and (or) the subcellular distribution of protein kinase dependent on cyclic AMP (adenosine 3':5'-monophosphate), since they influence cyclic-AMP-dependent responses to other hormones. Protein kinase activity was determined in rat liver homogenates and subcellular fractions, nuclear, large granular, microsomal and supernatant obtained by differential sedimentation in 0.25 M sucrose. 63% of the tissue protein kinase activity detected in absence of cyclic AMP reside in the particulate fractions. Upon addition of exogenous cyclic AMP, protein kinase activity is stimulated 1.8, 1.2, 1.2 and 4.5-fold in nuclear, large granular, microsomal and supernatant fractions, respectively. Under these conditions, 66% of tissue activity are found in the supernatant fraction. The activity sensitive to exogenous cyclic AMP resolves into a major (84%) cytosoluble and a minor (16%) nucleomicrosomal component. The latter activity resists elution with isotonic saline and is increased in the presence of Triton X-100. Three groups of rats were studied: control and adrenalectomized with or without cortisol treatment. In whole liver homogenates, both protein kinase activity detected in absence of exogenous cyclic AMP and sensitivity of the enzyme to cyclic AMP were comparable in all groups. Moreover, the distribution patterns of proteins kinase activity amoung the fractions were essentially the same in all groups of animals, whether or not particles had been treated with Triton X-100. Finally, in cell-free experiments, glucocorticoids alone or in combination with their intracellular receptor did not modify protein kinase activity of rat liver. Thus the results reported do not support the possibility that glucocorticoids influence cyclic AMP-dependent protein kinase in rat liver. Yet, this study provides data, not available before, on subcellular distribution of this enzyme in rat liver.     

Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.     

Characterization of calf-ovary adenosine;3':5'-monophosphate-dependent protein kinases and adenosine-3':5'-monophosphate-binding proteins.

Protein phosphokinase activity from the calf ovary cytosol (105000 X g supernatant fraction) has been resolved by chromatography and polyacrylamide gel electrophoresis into two major protein kinases, PK-H1 and PK-H2, both dependent on adenosine 3':5'-monophosphate (cyclic AMP). The enzymes have similar molecular weights (230000) and substrate specificities but differ in their cyclic-AMP-dependency and stimulation by cyclic AMP. The differences have been explained by the presence in PK-H1 of a unique cyclic-AMP-binding protein which has little catalytic activity associated with it. The cyclic-AMP-binding protein has a high affinity for cyclic AMP and in addition is able to inhibit the activity of the isolated catalytic subunit. The ovarian cyclic-AMP-dependent protein kinases have properties similar to those found in other tissues. They can be dissociated into catalytic and regulatory subunits and are inhibited by a heat-stable protein inhibitor isolated from rabbit skeletal muscle. Preincubation of the cytosol with high levels of cyclic AMP resulted in additional cyclic-AMP-dependent protein kinases and cyclic-AMP-binding proteins which include protein kinases and binding proteins of greater than 400 000 molecular weight.     

The separation, properties and possible subunit composition of adenosine 3',5'-monophosphate-dependent protein kinases in brown adipose tissue.

Two 8.5-S protein kinases (ATP : protein phosphotransferase EC 2.7.1.37) and one 6.6-S protein kinase were purified 500--1000-fold from the acid-soluble fraction of brown adipose tissue. The catalytic properties of the kinases were similar. Each kinase was activated by cyclic AMP and had two components of cyclic AMP binding. In the presence of 200 nM cyclic AMP, undissociated kinase activity sedimented at 7.7 or 5.5 S. Free catalytic activity (3.2 S) could be detected but was unstable. Free regulatory units could not be detected. The 8.5-S protein kinase was dissociated by freezing and thawing to a 7.7-S variety with loss of the higher affinity component of binding. The 7.7-S kinase was sedimented through linear gradients of sucrose containing different concentrations of cyclic AMP. At each concentration, kinase activity lost from the holoenzyme peak (% of original) was identical with the amount of cyclic AMP bound at equilibrium (% oof maximum). Similar experiments on the 8.5-S kinase showed that the binding component with higher affinity was not associated with the release of catalytic activity. The results were consistent with the propostal that the kinases isolated contained one more cyclic AMP binding subunit than catalytic subunit (3 : 2 for 8.5 S and 2 : 1 for 6.6 S) and that this extra subunit was released to give an equal number of subunits of each type before catalytic activity was liberated.     

Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase.

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.     

Sulfhydryl group reactivity of adenosine 3',5'-monophosphate dependent protein kinase from bovine heart: a probe of holoenzyme structure.

The spectrophotometric titration of SH groups in adenosine 3',5'-monophosphate (cAMP) dependent protein kinase from bovine heart muscle with 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB) is described. The holoenzyme (R2C2) contains 16 SH groups, 12 of which react with DTNB in the native enzyme. The SH groups are distributed 2 per catalytic (C) and 4 per regulatory (R) subunit. The binding of cAMP to the holoenzyme or isolated R subunit prevents the reaction of one SH group per R subunit. Modification of SH groups, however, has only a small effect on cAMP binding to R. Reaction of the C subunit with DTNB results in less than 95% loss of catalytic activity. The kinetics of the DTNB reaction and the reversal of the inactivation process by treatment with dithiothreitol suggest that the inactivation is associated with SH group modification. Inactivation studies with the holoenzyme show that: (1) the R subunit inhibits DTNG inactivation of the C subunit in the absence of cAMP; (2) the rate of inactivation of the dephosphoholoenzyme in the presence of cAMP is considerably faster than that of the free catalytic subunit; and (3) the rate of inactivation of the phosphoholoenzyme in the presence of cAMP is faster than that of the C subunit but slower than the dephosphoholoenzyme. The results are interpreted as evidence for a significant interaction of the R and C subunits in the presence of saturating concentrations of cAMP. This interaction is modulated by the state of phosphorylation of R. To account for the inactivation data, a short-lived ternary complex containing R, C, and cAMP is postulated to be in rapid equilibrium with the subunits.     

Role of 3',5'-cyclic AMP in the control of nuclear protein kinase activity

The role of cAMP in the regulation of nuclear protein kinase activity was investigated. Acidic nuclear proteins prepared from rat liver nuclei were separated by phosphocellulose chromatography into four peaks of protein kinase activity and two peaks of cAMP-binding activity. A fraction which bound cAMP also inhibited the most active nuclear protein kinase, K IV, and the inhibition was diminished in the presence of 5 μM cAMP. Further support for the regulation of nuclear kinases by cAMP was obtained using a regulatory subunit prepared from rabbit muscle protein kinase. The muscle regulatory subunit markedly inhibited liver nuclear kinase activities. The addition of cAMP partially restored the activities.     

Activation of the particulate low Km phosphodiesterase of adipocytes by addition of cAMP-dependent protein kinase

The purified catalytic subunit (C) of cAMP-dependent protein kinase produced a 2-fold activation of the low Km phosphodiesterase in crude microsomes (P-2 pellet) of rat adipocytes. This activation was C subunit concentration-dependent, ATP-dependent, blocked by a specific peptide inhibitor, and lost if the C subunit was first heat denatured. The concentration of ATP necessary for half-maximal activation of the low Km phosphodiesterase was 4.50 +/- 1.1 microM, which was nearly the same as the known Km of C subunit for ATP (3.1 microM) using other substrates. The concentration of C subunit producing half-maximal activation of phosphodiesterase was 0.22 +/- 0.04 microM, slightly less than the measured concentration of total C subunit in adipocytes (0.45 microM). The activation of the low Km phosphodiesterase by C subunit was specific, since on an equimolar basis, myosin light chain kinase, cGMP-dependent protein kinase, or Ca2+/calmodulin-dependent protein kinase II did not activate the enzyme. The percent stimulation of phosphodiesterase by C subunit was about the same as that produced by incubation of adipocytes with a cAMP analog, and the enzyme first activated in vivo with the analog was not activated to the same extent (on a percentage basis) by in vitro treatment with C subunit. Treatment of the crude microsomes with trypsin resulted in transfer of phosphodiesterase catalytic activity from the particulate to the supernatant fraction, but the enzyme in the supernatant was minimally activated by C subunit, suggesting either loss or dislocation of the regulatory component. The C subunit-mediated activation of phosphodiesterase was preserved after either transfer of phosphodiesterase activity to the supernatant fraction by nonionic detergents or partial purification of the transferred enzyme. The present findings are consistent with the suggestion that protein kinase regulates the concentration of cAMP through phosphodiesterase activation and provide direct evidence that the mechanism of activation involves phosphorylation.     

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.     

Hormonal effects on fat cell adenosine 3′,5′-monophosphate dependent protein kinase

Fat cell extracts were electrophoresed on polyacrylamide gels to separate the regulatory subunit and holoenzyme species of protein kinase. Gels were incubated with cyclic [³H]AMP ([³H]cAMP) and washed, and the bound [³H]cAMP was estimated. The band of [³H]cAMP found closest to the origin (Peak I) was associated with cAMP-dependent protamine kinase activity. A seond [³H]cAMP peak (Peak II) also contained protamine kinase activity. Although the kinase activity of Peak II was much less than Peak I, more [³H]-cAMP was bound in Peak II than in Peak I. The [³H]cAMP peak furthest from the origin (Peak III) was devoid of kinase activity.Incubation of extracts with cAMP prior to electrophoresis diminished or abolished kinase activity in Peaks I and II. This incubation also decreased [³H]cAMP binding in Peaks I and II, and increased binding in Peak III. When extracts were incubated with [³H]cAMP before electrophoresis, essentially all of the radioactivity was found in Peak III. It was concluded that Peak I represents a holoenzyme form and that Peak III is composed of the regulatory subunits of this enzyme. Peak II may represent a relatively inactive holoenzyme form not previously described.Incubation of adipocytes with epinephrine resulted in a dose- and time-dependent decrease in Peak I and increase in Peak III, and insulin opposed these effects of epinephrine. After 1-min incubations with epinephrine, the decreases in Peak I or increases in Peak III correlated with increases in phosphorylase a activity, decreases in glycogen synthase I activity and changes in cAMP, both in the presence and absence of insulin. However, after incubation with epinephrine for more than 2 min in the presence of insulin, phosphorylase a activity did not correlate with cAMP, suggesting that factors other than the cyclic nucleotide mediate the effects of epinephrine and insulin.     

Conversion of bovine cardiac adenosine cyclic 3',5'-phosphate dependent protein kinase to a heterodimer by removal of 45 residues at the N-terminus of the regulatory subunit

The type II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase from bovine heart, consisting of a dimeric regulatory subunit and two catalytic subunits, was converted to a heterodimer by limited tryptic digestion. Loss of the tetrameric structure was accompanied by proteolysis of the regulatory subunit to a form with an apparent molecular weight of 45 000 vs. 52 000 for the native subunit. The proteolyzed subunit behaved as a monomer, in contrast to the dimeric native subunit. Amino acid sequence analysis established that proteolysis removed 45 residues at the N-terminus, indicating that these 45 residues constitute the dimerizing domain of this protein. The kinetic properties of this heterodimer were indistinguishable from those of the native tetramer: half-maximal kinase activation occurred at 48 nM cAMP with a Hill coefficient of 1.45, the regulatory subunit bound 1.5 equiv of cAMP with half-maximal binding occurring at 33 nM, and kinetics for dissociation of bound cAMP were biphasic, indicating the presence of two different binding sites. These observations suggest that residues 1-45 function only in the formation of dimers and that dimerization has little influence on other functional properties of the regulatory subunit. More extensive proteolysis cleaved the monomeric fragment at Lys-311. The fragments resulting from this second cleavage did not dissociate, and the complex inhibited the catalytic subunit in a cAMP-dependent manner.     

Separation of regulatory and catalytic subunits of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase(s) of rabbit skeletal muscle

The cyclic 3′, 5′-adenosine monophosphate-dependent (cAMP-dependent) protein kinase(s) from rabbit skeletal muscle has been separated into catalytic and regulatory subunits by affinity chromatography utilizing a casein-Sepharose column in the presence of cAMP. The isolated catalytic subunit manifests full activity in the absence of cAMP but its requirement for this nucleotide is regained when the enzyme is reconstituted by addition of the regulatory subunit. Evidence is presented for the existence of more than a single type of regulatory or cAMP-binding subunit in muscle.