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)

The activation of cyclic AMP-dependent protein kinase has been found to be the predominant mode by which cyclic AMP (cAMP) leads to alterations of a large variety of cellular functions. The activation of the kinase results in the release of the catalytic subunit which as the free enzyme possesses phosphotransferase activity for a variety of specific protein substrates. Using a sensitive and specific cytofluorometric technique we monitored the appearance of free catalytic subunit in Reuber H35 hepatoma cells in culture after incubation with N6-1'-O-dibutyryl-cyclic AMP (DBcAMP), 8-bromoadenosine-3':5'-cyclic monophosphate (8-BrcAMP), and glucagon. The cytochemical method employs the heat-stable inhibitor of the free catalytic subunit which has been conjugated to fluorescein isothiocyanate (F:PKI) and was validated as described in the companion paper (Fletcher and Byus. 1982. J. Cell Biol. 93:719-726). Here we studied the temporal and spatial kinetics of the free catalytic subunit following activation of cAMP-dependent protein kinase by increasing concentrations of DBcAMP,8-BrcAMP, and glucagon. Under similar conditions protein kinase activation was also assessed biochemically in H35 cell supernatants by assaying the protein kinase activity ratio. Incubation of the hepatoma cells with DBcAMP (0.1 mM) led to an increase in the activity ratio from 0.2 in control cultures to a value of nearly 1.0 within a 1- to 2-h period. During this same period using the F:PKI probe, a significant increase in cytoplasmic and nucleolar fluorescence indicative of the release of the free catalytic subunit was coincidentally observed. In contrast to the rapid appearance of catalytic subunit in the cytoplasm and nucleolus of the cell within 5-15 min of the addition of DBcAMP, discernible nucleoplasmic fluorescence did not occur until after 1 h. H35 cell cultures incubated with 8-BrcAMP (0.01-1.0 mM) exhibited a more rapid activation of the protein kinase measured cytochemically compared to the cells treated with DBcAMP. Cultures incubated with 8-BrcAMP had significantly increased cytoplasmic and nucleolar fluorescence compared to unstimulated cells within 1 min of the addition of the analogue and reached a maximal level within 15 min. By employing a microspectrophotometer a distinct dose-dependent increase in cellular fluorescence (i.e., free catalytic subunit) was observed as the concentration of 8-BrcAMP was increased from 0.01 to 1.0 mM at 1, 5, 15, and 60 min following stimulation. The addition of glucagon (10(-6) M) to the culture also led to the activation of cAMP-dependent protein kinase as determined by an increase in the activity ratio. This increase was paralleled throughout the incubation period by a marked elevation in cytoplasmic and nucleolar fluorescence. The results reported herein suggest that both cyclic nucleotide analogues and a polypeptide hormone lead to the activation of cAMP-dependent protein kinase in similar intracellular compartments in Reuber H35 hepatoma cells...
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PMID:Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. II. Temporal and spatial kinetics. 628 33

In ovo [32P] phosphoproteins were analyzed during meiotic maturation of Xenopus laevis oocytes. A phosphoprotein of 105,000-dalton was found to increase early (one hour) after progesterone induction of meiosis. The pure heat-stable inhibitor (PKI) of cAMP-dependent protein kinase, which induces maturation, was microinjected into oocytes. Again the early increase in the 105,000-dalton [32P] phosphoprotein occurred. The burst in protein phosphorylation, which takes place at the period of germinal vesicle breakdown, was quantitatively and qualitatively comparable in progesterone and PKI-stimulated oocytes. In order to confirm the inverse relationship between the 105,000 dalton [32P] phosphoprotein increase and cAMP-dependent protein kinase activity, purified C-subunit of the kinase has been microinjected into oocytes. C-subunit which inhibits maturation did not increase significantly the 105,000-dalton [32P] phosphoprotein whereas it increased the total level of in ovo phosphorylation. Enucleation experiments favour the localization of the 105,000-dalton protein in both the oocyte cytoplasm and nucleus. Furthermore the progesterone-induced increase in the phosphorylation of the 105,000-dalton protein was found in the cytoplasmic compartment after oocyte enucleation.
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PMID:Early increase of a 105,000-dalton phosphoprotein during meiotic maturation of Xenopus laevis oocyte. 629 1

Microinjection of cAMP-dependent protein kinase inhibitor (1.8 microM) increases the cAMP level of Xenopus oocyte. Its effect was observed in full-grown (stage VI) as well as in vitellogenic (stage IV) oocytes. In contrast the inhibitor I1 of protein phosphatase-1 blocks cAMP accumulation. Progesterone (1 microM) decreases the cAMP level in control and in PKI-treated oocytes of both stages. These results show that cAMP concentration is regulated by a cAMP-dependent phosphorylation indicating the presence of a feedback mechanism. The feedback control is disrupted when oocyte is induced to mature by progesterone.
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PMID:cAMP-dependent protein kinase regulates in ovo cAMP level of the Xenopus oocyte: evidence for an intracellular feedback mechanism. 630 83

Amylase release from parotid acinar cells is a typical model of cAMP-mediated exocytosis. To obtain unequivocal data concerning the role of cAMP-dependent protein kinase (PKA) in amylase exocytosis, we undertook the direct introduction of the PKA catalytic subunit into the parotid acini by permeabilization with streptolysin O (SLO). In the presence of 100 hemolytic units/ml SLO, cAMP increased amylase release in a time- and dose-dependent manner. PKI-(5-24)-peptide, a specific PKA inhibitor, markedly inhibited amylase release, but the extent of inhibition was approximately 50%. On the other hand, the PKA catalytic subunit highly purified from bovine hearts significantly induced amylase release. The release was strictly dependent on the presence of SLO and the catalytic activity of PKA added. The catalytic subunit dose dependently induced amylase release, but the heat-inactivated subunit had no stimulatory effect. PKI-(5-24)-peptide completely blocked amylase release evoked by the subunit. These results clearly demonstrate that the catalytic subunit of PKA regulates cAMP-mediated amylase release through phosphorylation of unidentified protein(s) directly or indirectly involved in the process of exocytosis.
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PMID:Catalytic subunit of protein kinase A induces amylase release from streptolysin O-permeabilized parotid acini. 752 Sep 11

Whole-cell patch-clamp recordings from Vicia faba mesophyll protoplasts reveal that outward K+ current is increased in a dose-dependent fashion by intracellular application of cAMP. The enhancement of the outward current by cAMP is specific and it cannot be mimicked by a series of nucleotides that includes AMP, cGMP, and GMP. The enhancement is evoked by micromolar concentrations of cAMP in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine. PKI or Walsh inhibitor, a specific peptide inhibitor of cAMP-dependent protein kinase (PKA), inhibits the outward K+ current. Adenosine 3',5'-phosphothioate, a competitive inhibitor of PKA, has a similar effect. Conversely, the catalytic subunit of PKA (cAMP independent) from bovine brain enhances the magnitude of the outward K+ current in the absence of added cAMP. Our results indicate that cAMP modulates K+ channel activity in mesophyll cells and suggest that this modulation occurs through a cAMP-regulated protein kinase.
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PMID:Cyclic AMP stimulates K+ channel activity in mesophyll cells of Vicia faba L. 752 28

Regulation of L-type Ca2+ channel current [ICa(L)] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. ICa(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Stimulated ICa(L) (by 2 mumol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 mumol/L). When ICa(L) was enhanced by Bay K8644 (1 mumol/L), the enhanced basal ICa(L) was also reduced by PK-G. Basal ICa(L) (nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n = 36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG-PKI, 300 mumol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated ICa(L) (7 of 8 cells). When a holding potential of -80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca2+ channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca2+ channels at two separate sites.
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PMID:cGMP-dependent protein kinase regulation of the L-type Ca2+ current in rat ventricular myocytes. 755 27

The regulation of cardiac Cl- conductance by cAMP-dependent protein kinase (PKA) and cellular phosphatases was studied in isolated guinea pig ventricular myocytes by using wide-tipped, perfused pipettes to record whole-cell currents. Exposure to forskolin (Fsk) or isoproterenol (Iso) elicits a Cl- conductance that results exclusively from PKA-dependent phosphorylation because it can be completely abolished, or its activation fully prevented, by switching to pipette solution containing PKI, a synthetic peptide inhibitor of PKA. The Cl- conductance activated by micromolar concentrations of either agonist reached its steady-state amplitude in 1-2 min and was deactivated promptly and entirely, usually within 2 min, upon washing out the agonist, implying a continuous high level of activity of endogenous protein phosphatases. Accordingly, intracellular application of okadaic acid or microcystin, both potent inhibitors of protein phosphatases 1 and 2A, during exposure to Fsk enhanced the steady-state Cl- conductance and slowed its deactivation after washing out the Fsk. Maximal potentiation of the conductance, by approximately 60%, was obtained with pipette concentrations of approximately 10 microM okadaic acid (or approximately 5 microM microcystin) and did not result from an increase in the apparent affinity for Fsk. In the presence of maximally effective concentrations of okadaic acid and/or microcystin, deactivation of the enhanced Cl- conductance upon washout of agonist was incomplete, with about half of the conductance persisting indefinitely. That residual conductance did not reflect continued action of PKA because it was insensitive to PKI, but was identified as a fraction of the activated Cl- conductance by its biophysical characteristics. The results suggest that complete deactivation of the PKA-regulated cardiac Cl- conductance requires dephosphorylation by a type 1 and/or 2A phosphatase, but that partial deactivation can be accomplished by activity of some other phosphatase(s). These findings are consistent with sequential phosphorylation of a protein, probably the Cl- channel itself, at two different kinds of sites. The resulting phosphoproteins can be distinguished on the basis of their different contributions to whole-cell Cl- conductance.
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PMID:Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart. 768 43

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild-type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke-deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP-mediated mechanism.
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PMID:Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase. 779 20

The phosphorylation of substrate peptides derived from PKI, the heat-stable inhibitor protein of the cAMP-dependent protein kinase (PKA), has been studied with both PKA and the cGMP-dependent protein kinase (PKG) using a variety of substitution and deletion analogs. On the basis of Km, kcat and kcat/Km values, (Ser21)PKI alpha(14-22) amide (numbering based upon native PKI alpha) is the most effective peptide substrate yet discovered for either kinase, although other peptides, while phosphorylated considerably less efficiently by PKG, are more specific. Although the inhibitory peptide corresponding to this sequence (i.e., with an Ala at position 21) is a much more potent inhibitor of PKA than of PKG (approximately 250-fold), PKG actually exhibits a 60% higher kcat than does PKA with the (Ser21)PKI alpha(14-22) amide substrate peptide, with only a 20-fold higher Km value. The two key PKI residues within this peptide which were found to be essential for substrate activity with both kinases were Arg18 (P-3) and Ile22 (P+1). The Arg19 (P-2) residue, which contributes significantly to both PKI-based peptide inhibitors and substrates of PKA, was only a more minor contributor to PKG substrate efficacy. Of particular note, the Phe10 (P-11) residue, which contributes very substantially to high affinity binding of both PKI and longer PKI peptide inhibitors, neither positively nor negatively affects the kinetics of either PKA or PKG with PKI-based substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Heat-stable inhibitor protein derived peptide substrate analogs: phosphorylation by cAMP-dependent and cGMP-dependent protein kinases. 781 46

The signaling pathways mediating relaxation by vasoactive intestinal peptide (VIP), peptide histidine-isoleucine amide (PHI), isoproterenol (ISO), and sodium nitroprusside (SNP) were examined in dispersed rabbit and guinea pig gastric muscle cells. In rabbit muscle cells, SNP stimulated only guanosine 3',5'-cyclic monophosphate (cGMP) and cGMP-dependent protein kinase (cG-kinase) activity; VIP stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP, and both cG-kinase and cAMP-dependent protein kinase (cA-kinase) activities; PHI and ISO stimulated only cAMP and cA-kinase activity, and at higher concentrations, cross-activated cG-kinase. All four agents elicited concentration-dependent relaxation. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1 microM) selectively inhibited cA-kinase activity and abolished relaxation when only cA-kinase was elevated. 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c,d,e)- trinden-1-one (KT-5823; 1 microM) selectively inhibited cG-kinase activity and abolished relaxation when only cG-kinase was elevated. When both kinases were elevated, H-89 and KT-5823 partially inhibited relaxation and abolished relaxation in combination. In permeabilized guinea pig and rabbit muscle cells, all agents elicited relaxation and inhibited inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Both functions were inhibited in parallel fashion by protein kinase inhibitor PKI(6-22) and by KT-5823. We conclude that cA-kinase and cG-kinase act separately and in concert to inhibit IP3-dependent Ca2+ release and induce relaxation.
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PMID:Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells. 784 Jan 45


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