Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Temporal cellular events responsible for hormonal activation of responses mediated by the cAMP-dependent protein kinase (PKA) have been studied in living cells. By selectively perturbing molecular function of Gs, the catalytic subunit of PKA (C), or the nuclear factor CREB, in cells through microinjection of inhibitory agents specific for these molecules or activated forms of these molecules, we have obtained evidence for a requirement for the function of each of these molecules in the hormonal stimulation of cAMP-regulated genes. Moreover, by introducing fluorescently labeled PKA subunits into these cells as molecular tracers, or by immunofluorescence of C subunit, we have observed biological translocation of C subunit from the cytoplasm to the nucleus during transcriptional activation and a quenching of this by the inhibitor molecule, PKI. The implications of these cellular and molecular events in the signal transduction of hormonal responses are discussed.
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PMID:Signal transduction through the cAMP-dependent protein kinase. 793 49

Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was approximately 100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to reactivation. The cytosol-free models retained > 90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase.
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PMID:Role of cAMP in the reactivation of demembranated ram spermatozoa. 802 Jan 7

Cyclic GMP-dependent protein kinase (cGPK) activity was determined in rat pulmonary microvascular endothelial cells (RPMVEC) using cGMP-stimulated phosphorylation of BPDEtide and histone F2B substrates in the presence of PKI [peptide inhibitor of cAMP-dependent protein kinase (cAPK)]. RPMVEC cGPK activity was localized to the 100,000 x g cytosolic fraction. The EC50 for cGMP activation in the presence of PKI was 0.16 microM and H-89 inhibition under similar conditions showed an IC50 value of 0.16 microM. Anion-exchange chromatography of RPMVEC and rat lung cytosolic fractions showed separation of the cGMP-dependent from the cGMP-independent protein kinase activity and similar elution conductivities. Further, Western blots of RPMVEC active DEAE-Trisacryl fractions showed immunoreactivity using bovine Type I cGPK antiserum. Preliminary studies reveal six potential substrates phosphorylated by cGPK in RPMVEC. These studies describe an endothelial cell (EC) cGMP-receptor, cGPK, in addition to cGMP-activated (Type II) phosphodiesterase (PDE).
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PMID:Cyclic GMP-dependent protein kinase activity in rat pulmonary microvascular endothelial cells. 804 44

Relaxin (RLX), a reproductive hormone of the insulin family, increases heart rate in experimental animals. The cellular and ionic mechanisms responsible for this positive chronotropic effect remain unknown. We have investigated the actions of RLX on the action potential and underlying transmembrane ionic currents in single sinoatrial node cells of the rabbit heart under whole-cell voltage-clamp conditions, using both nystatin-perforated-patch and membrane-ruptured techniques. In this preparation RLX (0.8 to 80 nmol/L) caused reversible increases in the rate of spontaneous action potentials and a dose-dependent increase in the L-type calcium current, ICa(L). The best-fit Langmuir relation for the augmentation of ICa(L) yielded a threshold concentration of 1 nmol/L and a KD of 14 nmol/L. These effects of RLX appear to be mediated by increases in intracellular cyclic AMP (cAMP), since RLX was without effect after application of (1) the beta-adrenergic agonist isoprenaline (1 mumol/L) or (2) superfusion of the intracellular second messenger cAMP (100 mumol/L) or 8-Br-cAMP (100 to 200 mumol/L). Internal dialysis with an inhibitor of cAMP-dependent protein kinase (PKI, 7 mumol/L) abolished the effects of RLX. These results provide the first electrophysiological evidence that RLX modulates heart rate and contractility by increasing ICa(L) and suggest that the biochemical mechanism involves the formation of cAMP and activation of cAMP-dependent protein kinase.
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PMID:Relaxin increases heart rate by modulating calcium current in cardiac pacemaker cells. 811 61

The aim of this study was to evaluate the rapid regulation of cell-cell communication by using the microinjection of purified cAMP-dependent protein kinase (protein kinase A), the Ca(2+)-phospholipid-dependent protein kinase (protein kinase C), or the inhibitor proteins (PKI and CKI) that are, respectively, specific for each of these enzymes. Gap junction phenotypes of myometrial tissue and cells were studied by means of immunocytochemistry with antibody to connexin 43 (alpha 1; Cx43). Cells were enzymatically disaggregated from myometrium of nonpregnant, mid-pregnant (Day 14), and late-pregnant (Day 29) rabbit uteri (n = 8 per group) and seeded at high density such that after 4 days, cultures had the appearance of a cross-sectioned myometrium. Purified proteins and their subunits were microinjected, and intercellular communication was evaluated by monitoring Lucifer Yellow dye transfer. Cultures were treated with 0.5 mM 8Br-cAMP (8-bromo adenosine 3',5' cyclic monophosphate) or 10 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), which, respectively, activate protein kinase A and protein kinase C. Immunoreactive Cx43 and cell-cell communication were examined 5 min to 2 h later. Cx43 was detected in myometrial cryosections and cultured cells by indirect immunofluorescence, and its expression increased with gestation. Exposure to 8Br-cAMP increased the amount of immunoreactive Cx43. Basal dye transfer was minimal in nonpregnant cells, increased in cells of mid-pregnant uteri, and was maximal in late-pregnant cells. Treatment with 8Br-cAMP enhanced transfer in mid- and late-pregnant cells but had no obvious effect on cells from nonpregnant animals. OAG treatment inhibited dye transfer in greater than 95% of the cells tested irrespective of pregnancy status. PKI inhibited cell-cell communication within 2 min and up to 40 min. Injection of free catalytic subunit of protein kinase A following PKI inhibition restored communication within 2-3 min, with maximal transfer in 4-5 min. Protein kinase C inhibited communication, which resumed in < 3 min after injection of CKI. We conclude that rabbit myometrial cells engage in Cx43-mediated cell-cell communication and that this process increases during pregnancy. Further, activators of protein kinase A or injected free catalytic subunit rapidly enhances cell-cell communication, whereas activators of protein kinase C or the enzyme itself diminishes this process.
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PMID:Regulation of cell-cell communication mediated by connexin 43 in rabbit myometrial cells. 814 55

In teleost retinas, rods elongate in the light and shorten in the dark. Rod motility is mediated by the actin cytoskeleton of the inner segment and is regulated by cyclic AMP- or cyclic GMP-stimulated phosphorylation of target proteins. In this study, we have identified the target proteins of cyclic nucleotide-dependent kinases in rods, using preparations of isolated, motile rod inner-outer segments (RIS-ROS). Five proteins found in Percoll-purified RIS-ROS were phosphorylated in the presence of cAMP (> 10 nM), cGMP (> or = 10 microM) and exogenous catalytic subunit of cAMP-dependent protein kinase (PKA). The PKA inhibitor, PKI, blocked stimulation of phosphorylation by both cAMP and cGMP. Three cAMP-stimulated phosphoproteins were detected in cytoskeletal fractions of light- and dark-adapted RIS-ROS. One of these, PP33, appears to be a fish homologue of mammalian phosducin, based on immunolabeling by two different antibodies against mammalian phosducin and on electrophoretic characteristics in 2-D gels. Two additional phosducin immunoreactive bands were detected in Western blots. One, at 35 kDa, comigrated with a second cAMP-stimulated RIS-ROS phosphoprotein, PP35, which was also detected in the cytoskeleton. The other, at 37 kDa, was present in whole teleost retinas but not in purified RIS-ROS. Our results suggest that the effects of both cAMP and cGMP on teleost rod motility are mediated through PKA modulation of target phosphoproteins. These phosphoproteins include a cytoskeleton-associated phosducin homologue.
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PMID:Identification of cyclic nucleotide-regulated phosphoproteins, including phosducin, in motile rod inner-outer segments of teleosts. 815 21

MyoD is a nuclear phosphoprotein that belongs to the family of myogenic regulatory factors and acts in the transcriptional activation of muscle-specific genes. We have investigated the role of cAMP-dependent protein kinase (A-kinase) in modulating the nuclear locale of MyoD. Purified MyoD protein microinjected into the cytoplasm of rat embryo fibroblasts is rapidly translocated into the nucleus. Inhibition of A-kinase activity through injection of the specific inhibitory peptide PKI prevents this nuclear localisation. This inhibition of nuclear location is specifically reversed by injection of purified A-kinase catalytic subunit, showing the requirement for A-kinase in the nuclear import of MyoD. Site-directed mutagenesis of all the putative sites for A-kinase-dependent phosphorylation on MyoD, substituting serine or threonine residues for the non-phosphorylatable amino acid alanine, had no effect on nuclear import of mutated MyoD. These data exclude the possibility that the effect of A-kinase on the nuclear translocation of MyoD is mediated by direct phosphorylation of MyoD and imply that A-kinase operates through phosphorylation of components involved in the nuclear transport of MyoD.
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PMID:Nuclear import of the myogenic factor MyoD requires cAMP-dependent protein kinase activity but not the direct phosphorylation of MyoD. 820 83

The catalytic (C) subunit of cAMP-dependent protein kinase interacts with two classes of inhibitors. The regulatory (R) subunits, types I and II, associate to form an inactive holoenzyme complex that is activated in response to cAMP. The C-subunit is also inhibited by small heat-stable protein kinase inhibitors (PKI's). Inhibition by both PKI and RI-subunit requires the synergistic high-affinity binding of MgATP. The stabilizing effect of ATP was quantitated by using analytical gel chromatography. Both the type I holoenzyme and the C.PKI complex in the presence of MgATP show apparent Kd's for subunit association that are below 0.1 nM, while in the absence of MgATP the apparent Kd's are 125 nM and 2.3 microM, respectively, for the two complexes. In the absence of MgATP both complexes also can be dissociated readily and, hence, activated by salt-induced dissociation. Under physiological salt concentrations, salt-induced dissociation would be substantial in the absence of the high-affinity binding of MgATP. In both complexes, the ATPase activity of the free C-subunit is abolished. The off rates for MgATP also indicate that the type I holoenzyme is more stable than the C.PKI complex. The off rate (t1/2) for MgATP from the C.PKI complex is 17 min, while the off rate for the type I holoenzyme is 11.7 h. When the C.PKI complex is incubated with RI-subunit in the presence or absence of MgATP, the C-subunit preferentially reassociates with the RI-subunit, forming holoenzyme. In contrast, free PKI cannot compete for the C-subunit when it is part of a holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase: effect of MgATP on protein-protein interactions. 826 80

The catalytic (C) subunit of cAMP-dependent protein kinase is inhibited by the regulatory (R) subunit and by a thermostable inhibitor (PKI). Both inhibitors also affect the intracellular distribution of the C subunit. Whether injected into the cytoplasm or into the nucleus, free C subunit can enter and exit the nucleus freely. After 30 min its distribution is identical and is independent of the initial site of injection. In contrast, when C is injected into the cytoplasm complexed with R or PKI, the complexes are restricted to the cytoplasm (1-3). However, unlike the R subunit, which is restricted to the cytoplasm like the holoenzyme, free PKI enters the nucleus rapidly following its injection into the cytoplasm. When holoenzyme is injected directly into the nucleus, it cannot exit and return to the cytoplasm. In contrast, nuclear injection of a C.PKI complex results in the rapid exit of the C subunit from the nucleus. In equilibrated cells previously injected with the C subunit, subsequent cytoplasmic injection of either PKI or type 1 R depletes the nucleus of C although PKI does so faster, consistent with its ability to enter the nucleus. Both inhibitors block the cAMP response element-regulated gene expression. Hence PKI may serve as a nuclear scavenger of C providing a mechanism not only for inhibition but also for subcellular localization in the presence of cAMP by restricting the access of the C subunit to the nucleus.
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PMID:Thermostable inhibitor of cAMP-dependent protein kinase enhances the rate of export of the kinase catalytic subunit from the nucleus. 830 May 97

The crystal structure of the porcine heart catalytic subunit of cAMP-dependent protein kinase in a ternary complex with the MgATP analogue MnAMP-PNP and a pseudosubstrate inhibitor peptide, PKI(5-24), has been solved at 2.0 A resolution from monoclinic crystals of the catalytic subunit isoform CA. The refinement is presently at an R factor of 0.194 and the active site of the molecule is well defined. The glycine-rich phosphate anchor of the nucleotide binding fold motif of the protein kinase is a beta ribbon acting as a flap with conformational flexibility over the triphosphate group. The glycines seem to be conserved to avoid steric clash with ATP. The known synergistic effects of substrate binding can be explained by hydrogen bonds present only in the ternary complex. Implications for the kinetic scheme of binding order are discussed. The structure is assumed to represent a phosphotransfer competent conformation. The invariant conserved residue Asp166 is proposed to be the catalytic base and Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is functionally replaced by an Arg displaced by two residues in the primary sequence, suggesting invariance in three-dimensional space. The structure supports an in-line transfer with a pentacoordinate transition state at the phosphorus with very few nuclear movements.
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PMID:Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5-24). 838 54


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