In situ reassociation of the regulatory and catalytic subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase isoenzymes in AtT20 cells

Dissociation and reassociation of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases I and II were studied in intact AtT20 cells. Cells were stimulated with 50 microM forskolin to raise intracellular cAMP levels and induce complete dissociation of R and C subunits. After the removal of forskolin from the incubation medium cAMP levels rapidly declined to basal levels. Reassociation of R and C subunits was monitored by immunoprecipitation of cAMP-dependent protein kinase activity using anti-R immunoglobulins. The time course for reassociation of R and C subunits paralleled the loss of cellular cAMP. Total cAMP-dependent protein kinase activity and the ratio of protein kinase I to protein kinase II seen 30 min after the removal of forskolin was the same as in control cells. Similar results were seen using crude AtT20 cell extracts treated with exogenous cAMP and Mg2+. Our data showed that after removal of a stimulus from AtT20 cells inactivation of both cAMP-dependent protein kinase isoenzymes occurred by the rapid reassociation of R and C subunits to form holoenzyme. Our studies also showed that half of the type I regulatory subunit (RI) present in control cells contained bound cAMP. This represented approximately 30% of the cellular cAMP in nonstimulated cells. The cAMP bound to RI was resistant to hydrolysis by cyclic nucleotide phosphodiesterase but was dissociated from RI in the presence of excess purified bovine heart C. The RI subunits devoid of C may function to sequester cAMP and, thereby, prevent the activation of cAMP-dependent protein kinase activity in nonstimulated AtT20 cells.     

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.     

Enhanced activation of cAMP-dependent protein kinase by rapid synthesis and degradation of cAMP

Activation of cAMP-dependent protein kinase II by static and dynamic steady-state cAMP levels was studied by reconstituting an in vitro model system composed of hormone-sensitive adenylate cyclase, cyclic nucleotide phosphodiesterase, and cAMP-dependent protein kinase II. The rates of cAMP synthesis were regulated by incubating isolated membranes from AtT20 cells with various concentrations of forskolin. In the presence of 3-methylisobutylxanthine, the rate of protein kinase activation was proportional to the rate at which cAMP was synthesized, and there was a direct relationship between the degree of activation and the level of cAMP produced. The activation profiles of protein kinase generated in the presence of exogenous cAMP or cAMP produced by activation of adenylate cyclase in the absence of cAMP degradation were indistinguishable. Dynamic steady-state levels of cAMP were achieved by incubating the membranes with forskolin in the presence of purified cyclic nucleotide phosphodiesterase. Under these conditions, the apparent activation constant of protein kinase II for cAMP was reduced by 65-75%. This increased sensitivity to activation by cAMP was seen when phosphotransferase activity was measured directly in reaction mixtures containing membranes, protein kinase, and histone H2B or when regulatory and catalytic subunits were first separated by immunoprecipitation of holoenzyme and regulatory subunits with specific anti-serum. Our results are consistent with the hypothesis that rapid cAMP turnover may function as a mechanism for amplifying hormonal signals which use the cAMP-dependent protein kinase system.     

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells.

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.     

Coelution of the type II holoenzyme form of cAMP-dependent protein kinase with regulatory subunits of the type I form of cAMP-dependent protein kinase

The types and subunit composition of cAMP-dependent protein kinases in soluble rat ovarian extracts were investigated. Results demonstrated that three peaks of cAMP-dependent kinase activity could be resolved using DEAE-cellulose chromatography. Based on the sedimentation of cAMP-dependent protein kinase and regulatory subunits using sucrose density gradient centrifugation, identification of 8-N3[32P]cAMP labeled RI and RII in DEAE-cellulose column and sucrose gradient fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Scatchard analysis of the cAMP-stimulated activation of the eluted peaks of kinase activity, the following conclusions were drawn regarding the composition of the three peaks of cAMP-dependent protein kinase activity: peak 1, eluting with less than or equal to 0.05 M potassium phosphate, consisted of the type I form of cAMP-dependent protein kinase; peak 2, eluting with 0.065-0.11 M potassium phosphate, consisted of free RI and a type II tetrameric holoenzyme; peak 3, eluting with 0.125 M potassium phosphate, consisted of an apparent RIIC trimer, followed by the elution with 0.15 M potassium phosphate of free RII. The regulatory subunits were confirmed as authentic RI and RII based upon their molecular weights and autophosphorylation characteristics. The more basic elution of the type II holoenzyme with free RI was not attributable to the ionic properties of the regulatory subunits, based upon the isoelectric points of photolabeled RI and RII and upon the elution location from DEAE-cellulose of RI and RII on dissociation from their respective holoenzymes by cAMP. This is the first report of a type II holoenzyme eluting in low salt fractions with free RI, and of the presence of an apparent RIIC trimer in a soluble tissue extract.     

Sphingosine activates protein kinase A type II by a novel cAMP-independent mechanism

Protein kinase A (PKA) has long been recognized as playing a major role in many regulatory processes in cells through its activation by the ubiquitous second messenger cAMP. We show here a novel mode of activation of PKA type II that is independent of cAMP and is, instead, dependent on sphingosine. PKA type II is specifically activated by sphingosine and its analog, dimethylsphingosine, but not by sphingosine-1-phosphate or other lipids. Like cAMP, sphingosine activates PKA holoenzyme but not the catalytic subunit alone, suggesting that the activation is mediated by the regulatory subunits. However, sphingosine-activated PKA, but not cAMP-activated PKA, is inhibited by phosphatidylserine, suggesting a distinct mechanism of activation. Furthermore, unlike cAMP, sphingosine does not induce the dissociation of PKA holoenzyme into catalytic and regulatory subunits. Modulation of sphingosine levels in vivo results in alteration in basal membrane-associated PKA activity consistent with a direct effect of membrane sphingosine on PKA type II. Importantly, sphingosine-dependent but not cAMP-dependent activation of PKA specifically phosphorylates Ser58 of the multifunctional adapter protein 14-3-3zeta, promoting the conversion of dimeric 14-3-3 to a monomeric state, thus potentially modulating several biological functions. These results define a new mode of PKA activation that is sphingosine-dependent and mechanistically different from the classical cAMP-dependent activation of PKA. Furthermore, they suggest that stimuli that induce sphingosine accumulation and modulate phospholipid content at the cell membrane have the potential to activate PKA, thereby inducing the phosphorylation of distinct substrates and biological activities.     

Regulation of gene expression by transfected subunits of cAMP-dependent protein kinase

cAMP regulates the expression of several genes by activation of a promoter consensus sequence which functions as a cAMP-response element. Evidence indicated that this is accomplished via cAMP dissociation of cAMP-dependent protein kinase into its regulatory (R) and catalytic (C) subunits. Our investigations of the role of these two subunits in gene expression provide direct and quantitative evidence that the C subunit is required for cAMP stimulation of the cAMP-response element in the vasoactive-intestinal-peptide gene in rat pheochromocytoma cells. After cotransfection of a metallothionein-regulated C-subunit expression vector (pCEV) and a vasoactive-intestinal-peptide--chloramphenicol acetyltransferase construct containing a cAMP-response element, we could demonstrate expression of transfected C-alpha-subunit mRNA (truncated size 1.7 kb) by Northern blot and a concentration-dependent C subunit stimulation of chloramphenicol acetyltransferase activity. Basal activity was stimulated 12- and 50-fold by pCEV (30 micrograms), in the absence and presence, respectively, of Zn2+. Metallothionein-regulated expression of C was demonstrated by results that showed a 2-4-fold increase in chloramphenicol acetyltransferase activity in the presence versus the absence of 90 microM Zn2+. In contrast, overexpression of the R-II beta regulatory subunit did not stimulate chloramphenicol acetyltransferase activity, and R-II beta transfected together with C (ratio 2:1 and 4:1) inhibited the stimulation by the C subunit 70% and 90% respectively. Our results indicate that transfection of cAMP-dependent protein kinase subunits results in functional expression of both C-alpha and R-II beta subunits. Expression of the C subunit mediated cAMP-regulated gene expression but this expression could be inhibited by cotransfected R-II beta subunit, indicating intracellular reconstitution of the inactive holoenzyme of cAMP-dependent protein kinase.     

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 two mutants of the LLC-PK1 porcine kidney cell line affected in the catalytic subunit of the cAMP-dependent protein kinase

The catalytic (C) subunit activity of the cAMP-dependent protein kinase (cAMP-PK) from the mutant cell lines, FIB4 and FIB6, is only 10% compared with the parent cell line, LLC-PK1 [Jans and Hemmings (1986) FEBS Lett. 205, 127-131]. In order to understand the nature of the mutant phenotypes the cAMP-PK from parent and mutant cell lines was studied in more detail. Analysis of mutant cAMP-PK activity by ion-exchange chromatography revealed that kinase activity associated with type I holoenzyme of both FIB4 and FIB6 was only 5% parental, and the activity of the type II holoenzyme was about 20% parental. The type I regulatory (RI) subunits associated with the type I were also found to be reduced by 70-80% in both mutants, whereas the type II R subunit levels were similar to that of the parent. The residual kinase activity associated with the type I holoenzyme from FIB4 and FIB6 could not be activated by cAMP whereas the type II holoenzyme was activated by cAMP (Ka of 5.5 X 10(-8) M), and showed normal affinities for Kemptamide and ATP. A polyclonal antibody to the catalytic subunit was used to quantify the level of this protein in wild-type and mutant cells. This analysis showed that FIB4 and FIB6 had nearly normal levels of C subunit, suggesting that the C subunit synthesized by the mutants was mostly inactive. As both type I and type II cAMP-PK holoenzymes were abnormal, the most likely explanation of the mutant phenotype is a defect either in the structural gene for the C subunit or in an enzyme involved in its posttranslational processing. However, a second lesion affecting the RI subunit cannot be ruled out at this moment.     

Signaling the signal, cyclic AMP-dependent protein kinase inhibition by insulin-formed H2O2 and reactivation by thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.     

The consequences of introducing an autophosphorylation site into the type I regulatory subunit of cAMP-dependent protein kinase

The type I and type II regulatory subunits of cAMP-dependent protein kinase can be distinguished by autophosphorylation. The type II regulatory subunits have an autophosphorylation site at a proteolytically sensitive hinge region, while the type I regulatory subunits have a pseudophosphorylation site. Only holoenzyme formed with type I regulatory subunits has a high affinity binding site for MgATP. In order to determine the functional consequences of regulatory subunit phosphorylation on interaction with the catalytic subunit, an autophosphorylation site was introduced into the type I regulatory subunit using recombinant DNA techniques. When Ala97 at the hinge region of the type I regulatory subunit was replaced with Ser, the regulatory subunit became a good substrate for the catalytic subunit. Stoichiometric phosphorylation occurred exclusively at Ser97. Radioactivity was incorporated primarily into the recombinant regulatory subunit when catalytic subunit and [gamma-32P]ATP were added to the total bacterial extract. Phosphorylation of the mutant regulatory subunit also occurred readily following polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose. Phosphorylation occurred as an intramolecular event in the absence of cAMP indicating that the hinge region of the regulatory subunit occupies the substrate recognition site of the catalytic subunit in the holoenzyme complex. Holoenzyme formed with both the wild type and mutant regulatory subunits was susceptible to dissociation in the presence of high salt; however, only the native holoenzyme was stabilized by MgATP. In contrast to the wild type holoenzyme, the affinity of the mutant holoenzyme for cAMP was not reduced in the presence of MgATP. Holoenzyme formation also was not facilitated by MgATP.     

Effect of microinjections of cAMP-dependent protein kinase subunits on macromolecular syntheses in loach Misgurnus fossilis L. embryos

The level of cAMP and the effects of the regulatory and catalytic subunits of cAMP-dependent protein kinase of type II on protein, RNA and DNA synthesis in loach embryos were analysed. It was found that the injections of the catalytic subunit cause a sharp decrease in the rate of macromolecular synthesis while an increase in the concentration of this protein leads to the death of the embryo. Injections of the regulatory subunit result in marked stimulation of protein, RNA and DNA synthesis. The effect of the regulatory subunit on these processes is rather complex. Experiments on the transplantation of the nuclei from the embryos treated with the protein kinase subunits into normal embryos were carried out.     

Chromatographic behavior of cyclic AMP dependent protein kinase and its subunits from Dictyostelium discoideum

An adenosine cyclic 3',5'-monophosphate (cAMP) dependent protein kinase has recently been shown to exist in Dictyostelium discoideum and to be developmentally regulated. In this report we have followed the chromatographic behavior of both the holoenzyme and its subunits. A cAMP-dependent holoenzyme could be obtained from the 100000 g soluble fraction after passage through DE-52 cellulose (pH 7.5) and Sephacryl S300. Under conditions of low pH the holoenzyme could be further purified by flat-bed electrofocusing (pI = 6.8). Application of the holoenzyme to electrofocusing at high pH resulted in dissociation of the holoenzyme into a cAMP binding component (pI = 6.1) and a cAMP-independent catalytic activity (pI = 7.4). Dissociation of the holoenzyme into subunits also occurred during histone affinity chromatography and gel filtration chromatography (S300) in the presence of a dissociating buffer. Although the subunit structure was clearly evident during chromatography, the holoenzyme could not be dissociated by simple addition of cAMP to the extract. The catalytic subunit could be purified further by CM-Sephadex, DE-52 cellulose (pH 8.5), histone affinity, and hydrophobic chromatography. The regulatory subunit was further purified by DE-52 cellulose (pH 8.5) and cAMP affinity chromatography. Proof that the cAMP binding activity and the cAMP-independent catalytic activity were in fact the regulatory and catalytic subunits was shown by reconstitution of the cAMP-dependent holoenzyme from the purified subunits. By using these separation procedures, one can obtain from extracts of Dictyostelium the subunits that are free of each other as well as free of any endogenous protein substrates.     

Isolation and characterization of a dimeric cAMP-dependent protein kinase from the fungus Saccobolus platensis

A cAMP-dependent protein kinase from mycelia of Saccobolus platensis was characterized. The holoenzyme seems to be a dimer (i.e., regulatory subunit--catalytic subunit) of 78,000 Da, slightly activated by cAMP but susceptible to dissociation into its subunits by cAMP, or by kemptide and protamine, the best substrates for Saccobolus protein kinase. The regulatory subunit was purified to homogeneity by affinity chromatography. It is highly specific for cAMP and has two types of binding sites but failed to inhibit the phosphotransferase activity of the homologous or the heterologous (bovine heart) catalytic components. The activity of the catalytic subunit was completely abolished by the regulatory component of the bovine heart protein kinase as well as by a synthetic peptide corresponding to the active site of the mammalian protein kinase inhibitor. The data suggest that interaction between the subunits of the S. platensis protein kinase is different than that found in cAMP-dependent protein kinases from other sources. Similarities and differences between the Saccobolus protein kinase and enzymes from low eucaryotes and mammalian tissues are discussed.     

cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

A mechanistic and kinetic analysis of the interactions of the diastereoisomers of adenosine 3'',5''-(cyclic)phosphorothioate with purified cyclic AMP-dependent protein kinase.

The binding affinities of the diastereoisomers of adenosine 3',5'-(cyclic)phosphorothioate, Sp-cAMP[S] and Rp-cAMP[S], for the cyclic AMP- (cAMP-)binding sites on purified and reconstituted pig heart type II cAMP-dependent protein kinase holoenzyme were determined by measuring the ability of these compounds to displace [3H]cAMP from this enzyme. Sp-cAMP[S], a cAMP agonist, displaced 50% of the [3H]cAMP bound to the holoenzyme at a concentration 10-fold higher than that of cAMP; Rp-cAMP[S], a cAMP antagonist, required a 100-fold higher concentration relative to cAMP. Activation of the isolated holoenzyme, determined as phosphotransferase activity, was measured in the presence of the agonist and in the absence and in the presence of increasing concentrations of the antagonist. The results of fitting the activation data to sigmoid curves with a non-linear-regression program and to Hill plots by using a linear-regression program showed that Rp-cAMP[S] had no effect on Vmax, increased the EC50 values for agonist activation and had no effect on the co-operativity of activation (h). A Ki value of 11 microM was determined for Rp-cAMP[S] inhibition of cAMP-induced activation of purified type II cAMP-dependent protein kinase. Electrophoresis of the holoenzyme on polyacrylamide gels under non-denaturing conditions in the presence of saturating concentrations of the diastereoisomers resulted in 100% dissociation of the subunits with Sp-cAMP[S] and 0% dissociation with Rp-cAMP[S]. Sp-cAMP[S], the isomer with an axial exocyclic sulphur atom, binds to the holoenzyme, releases the catalytic subunit and activates the phosphotransferase activity. Rp-cAMP[S], the isomer with an equatorial exocyclic sulphur atom, binds to the holoenzyme but does not result in dissociation, and thus acts as a competitive inhibitor of phosphotransferase activity.     

Transcriptional activity in previtellogenic oocyte germinal vesicles from Xenopus laevis

Receptor interactions of parotid acinar cells with beta-agonists are mediated by cyclic 3',5'-monophosphate (cAMP) and expressed as cAMP-dependent protein kinase (cAPK) activation. In addition to its location in the cytoplasm, we have shown that cAPK is associated with the nuclear non-histone protein (NHP) fraction (0.35 M NaCl extract) of rat parotid acinar cells. Nuclei were prepared from isolated parotid acini with minimal contamination from other cell types or cytoplasmic components. The nuclear cAPK activity was inhibited by the thermostable inhibitor and was stimulated by the addition of exogenous cAMP to the assay, indicating that the enzyme is present in the holoenzyme form. Enzyme activity was not increased in the presence of detergent, suggesting that cAPK is not bound to the nuclear membrane. Photoaffinity-labeling studies with an 8-azido analog of cAMP showed that regulatory subunits of both type I and type II cAPK isozymes are present in parotid cell nuclei. Short-term in vitro stimulation of the acini with 10(-6) M isoproterenol did not alter cAPK activity in the nuclear fraction. These findings indicate that compartmentation of cAPK into nuclear and extranuclear locations in rat parotid acinar cells is similar to that of several other cell types which are responsive to hormonal stimulation.     

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.