Changes in cAMP-dependent protein kinase activity in the 10,000g sediment of sea urchin embryos during early development

cAMP-dependent protein kinase was found in the sediment obtained by centrifuging a homogenate of sea urchin embryos at 10,000g for 20 min, and was solubilized with 1% Triton X-100. This enzyme was eluted at 0.16 M NaCl in a linear concentration gradient on a DEAE-cellulose column, at which cAMP-dependent protein kinase found in the supernatant was also eluted. The enzyme activity was enhanced about 1.5-fold in the presence of 1 μM cAMP, and increased somewhat by adding cGMP or cIMP. The activation by cAMP of protein kinase in the sedimentable fraction was lower than in the supernatant fraction. The properties of the enzyme found in the 10,000g sediment and in the supernatant differ somewhat. The activity of the cAMP-dependent protein kinase in the 10,000g sediment was high in the embryos at the blastula, the swimming blastula, and the mesenchyme blastula stages. On the other hand, the activity was undetectable in unfertilized eggs and in embryos at the morula, the gastrula, and the pluteus stages.     

cAMP-dependent protein kinase in sea urchin embryos

cAMP-dependent protein kinase in the supernatant fraction of the homogenate of sea urchin eggs and embryos obtained by centrifugation at 105,000g was investigated in the present study. In the previous report, the dissociation constant between cAMP-binding proteins and cAMP changed during the development. This suggests that the nature of cAMP-dependent protein kinase, which has been well established to be the major cAMP receptor, changes during the development. In the present study, four protein kinases were separated through DEAE-cellulose column from the supernatant of unfertilized egg homogenate. One of them was cAMP-dependent protein kinase. The others were cAMP-independent ones. One among them was phosvitin kinase, and the others were not identified at present. The activity of cAMP-dependent protein kinase gradually increased during a period from fertilization to the swimming blastula stage. During this period, cleavages occurred at a high rate, and the rate decreased after hatching out. Thus, it is supposed that cAMP-dependent protein kinase in the supernatant may take a part in the mechanism of cleavage. The activity, however, became very low at the mesenchyme blastula, the gastrula, and the pluteus stages. cAMP-binding capacity was observed in the sedimentable fraction and the supernatant fraction, respectively, obtained by 105,000g centrifugation at all stages examined. If the structure-bound cAMP-binding protein is also cAMP-dependent protein kinase, it may play different roles in the mechanism of development.     

A low molecular weight substance purified from human placenta inhibits cAMP-dependent protein kinase and activates protein kinase C

We have purified from human placenta a low molecular mass substance that inhibits cAMP-dependent protein kinase and activates protein kinase C. This protein kinase regulator was purified in three steps: (1) homogenizing placentas in chloroform/methanol and extracting the regulator into water; (2) eluting a strong anion exchange high performance liquid chromatography (HPLC) column with a quaternary gradient; and (3) eluting a reversed-phase HPLC column with a binary gradient. The regulator was found to be highly purified by HPLC, thin-layer chromatography (TLC) and laser desorption ionization mass spectrometry with a molecular mass of 703 Daltons by the latter procedure. The physical and biochemical properties of this protein kinase regulator suggest that it is a phospholipid but it did not co-elute by HPLC or by TLC with any of the known phospholipid activators of protein kinase C.     

Altered cAMP-dependent protein kinase subunit immunolabeling in post-mortem brain from patients with bipolar affective disorder

Previous findings of reduced [3H]cAMP binding and increased activities of cAMP-dependent protein kinase (PKA) in discrete post-mortem brain regions from patients with bipolar affective disorder (BD) suggest that PKA, the major downstream target of cAMP, is also affected in this illness. As prolonged elevation of intracellular cAMP levels can modify PKA regulatory (R) and catalytic (C) subunit levels, we sought to determine whether these PKA abnormalities are related to changes in the abundance of PKA subunits in BD brain. Using immunoblotting techniques along with PKA subunit isoform-specific polyclonal antisera, levels of PKA RIalpha, RIbeta, RIIalpha, RIIbeta and Calpha subunits were measured in cytosolic and particulate fractions of temporal, frontal and parietal cortices of post-mortem brain from BD patients and matched, non-neurological, non-psychiatric controls. Immunoreactive levels of cytosolic Calpha in temporal and frontal cortices, as well as that of cytosolic RIIbeta in temporal cortex, were significantly higher in the BD compared with the matched control brains. These changes were independent of age, post-mortem interval or pH and unrelated to ante-mortem lithium treatment or suicide. These findings strengthen further the notion that the cAMP/PKA signaling system is up-regulated in discrete cerebral cortical regions in BD.     

Dipyridamole stimulates types II cAMP-dependent protein kinase in vitro

Dipyridamole activates in vitro type II CAMP-dependent protein kinase. This agent stimulates the autophosphorylation of the regulatory subunit in the presence of CAMP but not so in the absence of the cyclic nucleotide. The activation was also observed with exogenous substrates such as casein, histone 2A and MAP 2. This stimulation did not seem to be related to the cAMP binding to the R II subunit of the enzyme. Competition binding experiments showed that dipyridamole does not compete with adenosine for the A₁ receptor. The results suggest that the reported regulatory properties of dipyridamole on lipid metabolism (González-Nicolás et al. Int J Biochem 21: 883–888, 1989) might be mediated through a direct action — an activation — on the catalytic subunit of a cAMP-dependent protein kinase.     

High-performance liquid chromatographic identification and quantitation of cyclic adenosine 3':5'-monophosphate in higher (Phaseolus vulgaris L.) and lower (Chlorella sp.) plants

Cyclic adenosine 3':5'-monophosphate (cAMP) was extracted from Phaseolus vulgaris L. cv. Limburg seedlings and Chlorella sp. The cAMP was purified by charcoal adsorption, polyvinylpyrrolidone (PVP) and Dowex 50 W × 8 column chromatography and by preparative high-performance liquid chromatography (HPLC). Quantitation was achieved by combining phosphodiesterase (PDE) treatment with analytical HPLC (reversed phase ion-pair partition chromatography) and cAMP-dependent protein kinase activation. This provided a very specific, accurate and sensitive assay for cAMP determinations. The cAMP content found in Chlorella (70–90 pmol/g dry wt) was comparable with previous reports using other quantitation methods, whereas the endogenous concentration found in bean seedlings (92 pmol/g dry wt) was considerably lower than previously reported data.     

Role of cAMP-dependent protein kinase in the regulation of platelet procoagulant activity

The membrane microparticle (MP) formation and phosphatidylserine (PS) exposure evoked by platelet activation provide catalytic surfaces for thrombin generation. Several reports have indicated the effects of cAMP-elevating agents on agonist-induced MP formation and PS exposure; however, the mechanism still remains unclear. Here we show that inhibition of basal cyclic AMP-dependent protein kinase (PKA) activity incurred platelet MP formation and PS exposure. Pretreatment of platelets with cAMP-elevating agent, forskolin, abolished thrombin plus collagen-induced MP formation and PS exposure, and obviously decreased calcium ionophore-evoked MP shedding. Moreover, the inhibitory effects of forskolin on agonists-induced MP formation and PS exposure were reversed by the PKA inhibitor H89. PKA inhibitor-induced MP formation was dose-dependently inhibited by calpain inhibitor MDL28170, and forskolin abrogated thrombin plus collagen-induced calpain activation. In conclusion, PKA plays key roles in the regulation of platelet MP formation and PS exposure. PKA-mediated MP shedding is dependent on calpain activation.     

Analytical subcellular distribution of cAMP-dependent protein kinase activity in bull sperm

The distribution of the cAMP-dependent protein kinase activity in bull ejaculated sperm has been investigated. This activity proved to be mainly present in a soluble form inside the cell. Sperm fractionation into heads and flagellar fragments, using differential centrifugation or centrifugal elutriation, has shown that the particulate cAMP-dependent protein kinase activity was mainly associated with the flagellar structures. A much activity was shown to be associated with the head fraction. Some activity could also be detected in the purified plasma membrane fraction.     

Purification and characterization of a dimer form of the cAMP-dependent protein kinase from mouse liver cytosol

A protein kinase that phosphorylates histones and polysomal proteins was partially purified from mouse liver cytosol. The active enzyme has a molecular mass of 100 kDa and a phosphorylatable subunit of 54 kDa. Biochemical as well as immunological data suggest that the enzyme is a heterodimer composed of the catalytic subunit of cyclic AMP-dependent protein kinase and the R_II regulatory subunit. This RC form does not seem to dissociate upon activation with 3, 5 cyclic AMP and exhibits identical specificity as the classical cAMP-dependent protein kinase (2.7.1.37). The enzyme is affected by the 3, 5 cyclic phosphates of adenosine mainly, but also of guanosine, uridine and cytidine in a substrate-dependent manner. Cyclic nucleotides slightly stimulate phosphate incorporation into histones, while phosphorylation of polysomal proteins in intact polysomes is dramatically increased. The substrate- specific stimulatory effects of 3, 5 cyclic nucleotides are due to repression of the inhibition exerted upon the reaction, by negatively charged macromolecules such as RNA, DNA and to a lesser extent heparin.     

Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide-nucleoside conjugate AdcAhxArg6

Kinetic analysis of the inhibition of the phosphorylation of Kemptide, (LRRASLG), catalyzed by the catalytic subunit of cAMP-dependent protein kinase, by a peptide-nucleoside conjugate inhibitor AdcAhxArg6 was carried out over a wide range of ATP and peptide concentrations. A simple procedure was proposed for characterization of the interaction of this inhibitor with the free enzyme, and with the enzyme-ATP and enzyme-peptide complexes. The second-order rate constants, calculated from the steady-state reaction kinetics, were used for this analysis to avoid the complications related to the complex catalytic mechanism of the protein kinase catalyzed reaction.     

Evidence for the activation of 6-phosphofructo-1-kinase by cAMP-dependent protein kinase in Aspergillus niger

Abstract The change from pentose phosphate pathway to glycolysis plays a significant role in the physiology of Aspergillus niger during the induction of citric acid accumulation. Evidence is shown for the importance of 6-phophofructo-1-kinase in this process since it is activated by phosphorylation. By incubating a purified active form of enzyme together with commercially available alkaline phosphatase, 6-phosphofructo-1-kinase activity was lost after a certain time suggesting that the enzyme was dephosphorylated. Inactive 6-phosphofructo-1-kinase could be isolated from the cells in the early stage of growth in a high citric acid yielding medium. The enzyme was 'in vitro' activated by isolated protein kinase in the presence of cAMP, ATP and Mg²⁺ ions. Additional evidence for covalent phosphorylation of inactive 6-phosphofructo-1-kinase was obtained by incubating both enzymes together with labelled [ γ −³²P]ATP. The activating enzyme was partially purified from A. niger mycelium.     

Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.     

Characterization of the isolated cAMP-binding B domain of cAMP-dependent protein kinase.

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.     

Inhibition of 2'-phosphodiesterase by cAMP-dependent protein kinase. Involvement of phosphorylation of protein inhibitor

2'-Phosphodiesterase from NIH 3T3 cells was purified about 530-fold. Treatment of the cell lysate with the cAMP-dependent protein kinase causing the 2'-phosphodiesterase inhibition did not result in phosphorylation of the enzyme itself. The kinase was found to phosphorylate a specific 18-kDa protein, the phosphorylated form of this protein being the inhibitor of 2'-phosphodiesterase.     

Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase

The reaction mechanism of the catalytic phosphoryl transfer of cAMP-dependent protein kinase (cAPK) was investigated by semi-empirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol(-1) and the reaction itself to be exothermic by 12.2 kcal mol(-1). The active site residue Asp166, which was often proposed to act as a catalytic base, does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two-dimensional energy hypersurfaces of the protein kinase wild-type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.     

Activation of K+ channels in renal medullary vesicles by cAMP-dependent protein kinase

Summary ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide-and chloride-sensitive²²Na⁺ uptake and barium-sensitive, voltage-dependent⁸⁶Rb⁺-influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1mm ATP, and 50mm KCl, the barium-sensitive⁸⁶Rb⁺ influx increased from 361±138 to 528±120pm/mg prot · 30 sec (P<0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 g/ml) was also present in the vesicle solutions. The stimulation of⁸⁶Rb⁺ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.     

Different phosphorylation behaviour of regulatory subunit isoforms of type 11 cAMP-dependent protein kinase from bovine heart

Two forms of the regulatory subunit of the type II cAMP-dependent protein kinase (RII₅₅ and RII₅₂) were identified from bovine heart by gel electrophoretic behaviour. After autophosphorylation the RII₅₅ isoform migrated more slowly (RII_55/57) while the migration of RII₅₂ isoform did not shift. Both isoforms showed different affinity for cAMP. The RII_55/57 isoform was eluted from a cAMP-agarose column at 10 mM cAMP at low ionic strenght whereas the RII₅₂ isoform required cAMP, plus 2 M NaCl. Partial proteolysis, using trypsin or formic acid, of autophosphorylated regulatory subunit isoforms resulted in different cleavage pattern as determined by peptide mapping. However, the V8¹²⁵I-peptides patterns of both isoforms are quite similar.Incubation of partially purified holoenzyme with 10 nM [-³²P]ATP (low ATP concentration) yielded a single band of Mr = 57,000 which corresponds to the RII_55/57 isoform. The incubation, however, at 20 µM [-³²P]ATP yielded two phosphobands corresponding to both RII_55/57 and RII₅₂ isoforms. The phosphorylation of RII₅₂ took place with a lower efficiency and was more sensitive to the cAMP than the corresponding phosphorylation of the RII_55/57.     

Catalytic independent functions of a protein kinase as revealed by a kinase-dead mutant: study of the Lys72His mutant of cAMP-dependent kinase

A highly conserved lysine in subdomain II is required for high catalytic activity among the protein kinases. This lysine interacts directly with ATP and mutation of this residue leads to a classical "kinase-dead" mutant. This study describes the biophysical and functional properties of a kinase-dead mutant of cAMP-dependent kinase where Lys72 was replaced with His. Although the mutant protein is less stable than the wild-type catalytic subunit, it is fully capable of binding ATP. The results highlight the effect of the mutation on stability and overall organization of the protein, especially the small lobe. Phosphorylation of the activation loop by a heterologous kinase, 3-phosphoinositide-dependent protein kinase-1 (PDK-1) also contributes dramatically to the global organization of the entire active site region. Deuterium-exchange mass spectrometry (DXMS) indicates a concerted stabilization of the entire active site following the addition of this single phosphate to the activation loop. Furthermore the mutant C-subunit is capable of binding both the type I and II regulatory subunits, but only after phosphorylation of the activation loop. This highlights the role of the large lobe as a scaffold for the regulatory subunits independent of catalytic competency and suggests that kinase dead members of the protein kinase superfamily may still have other important biological roles although they lack catalytic activity.     

Activation of protein kinase isoenzymes under near physiological conditions. Evidence that both types (A and B) of cAMP binding sites are involved in the activation of protein kinase by cAMP and 8-N3-cAMP

cAMP-dependent protein kinase I and II (cAKI and cAKII) were incubated under near physiological conditions in the presence of various concentrations of 8-N3-c[3H]AMP or c[3H]AMP. Both types (A and B) of cyclic nucleotide binding sites of cAKI or cAKII were occupied to a similar extent and the degree of their occupation correlated with the degree of kinase activation. cAKI and cAKII bound cAMP in an apparent positively cooperative manner in the presence of Mg2+, ATP. 8-N3-c[3H]AMP dissociated several orders of magnitude faster from site A than site B of the regulatory moiety of cAKII, and was photo-incorporated only when bound to site B.