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)

We studied a signaling pathway for the activation of the superoxide (O2-)-generating NADPH oxidase and effects of cAMP on the pathway using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of protein kinase C (PKC) activator, phorbol myristate acetate (PMA), and a non-hydrolyzable GTP analogue, GTP gamma S in the presence of ATP and Mg2+. The O2- production by PMA not by GTP gamma S was inhibited by inhibitors of PKC. The production by PMA and GTP gamma S was inhibited by a GDP analogue, GDP beta S, in the same dose-dependent manner and the production by PMA was not enhanced by the addition of GTP gamma S and vice versa. These findings suggest the presence of a GTP-binding protein which follows PKC in the activation pathway. The O2- production by PMA and GTP gamma S was dose-dependently inhibited by cAMP and the inhibition was completely restored by an inhibitor of cAMP-dependent protein kinase, H-89, indicating that cAMP blocks the activating pathway at the site between the GTP-binding protein located downstream of PKC and the NADPH oxidase by activating cAMP-dependent protein kinase. The activation of the oxidase by sodium dodecyl sulfate (SDS) seemed to be different from the above pathway. It needed higher concentrations of GDP beta S for inhibition, did not absolutely need ATP and was inhibited by neither cAMP nor protein kinase C inhibitors. Moreover, the O2- production by the combination of GTP gamma S and SDS or of PMA and SDS was essentially the same as the sum of the production by each stimulant alone. We may conclude from the observations that the signaling pathway involving PKC for the activation of the oxidase is distinct from the pathway induced by SDS: the former is blocked by cAMP at the site between the GTP-binding protein located downstream of PKC and the oxidase and the latter is cAMP-insensitive.
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PMID:Cyclic AMP inhibits the respiratory burst of electropermeabilized human neutrophils at a downstream site of protein kinase C. 838 37

Rap1b, a member of the Ras superfamily of low molecular weight GTP-binding proteins, can be phosphorylated by cAMP-dependent protein kinase (protein kinase A). The experiments presented here were undertaken to determine the precise site of this phosphorylation. Because the Rap1 proteins are highly homologous, there are no specific antibodies able to discriminate between them. To overcome this problem, we used a transient expression system of a fused protein containing in the NH2 terminus an epitope for a known antibody. Using this system, the transfected protein was expressed at a high level and was localized in a perinuclear structure, as previously reported for the endogenous Rap1 proteins. The mutational analysis of Rap1b revealed Ser179 as the residue involved in the protein kinase A-mediated phosphorylation. The presence of a Lys179 instead of the wild-type Ser179 (resembling the Rap1a sequence) rendered Ser180 a better substrate for phosphorylation caused by protein kinase A. The mobility shift of Rap1b in SDS gels, observed in cells that were stimulated with agonists that increase cAMP, was caused, at least in part, by the phosphorylation of Rap1b.
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PMID:Mutational analysis of the cAMP-dependent protein kinase-mediated phosphorylation site of Rap1b. 846 83

Oxytocin increases myometrial intracellular free calcium by promotion of calcium entry and release of calcium from intracellular stores. Calcium release from intracellular stores is secondary to an increase in phosphoinositide (PI) turnover and generation of IP3. We have explored the biochemical basis for the coupling of oxytocin (OT) to phospholipase C (PLC). Rat myometrial membranes contain PLC beta, gamma, and delta isoforms as well as the GTP-binding proteins G alpha(q) and G alpha(11). Oxytocin stimulates both GTPase and PLC activity in rat and human myometrial membranes. These data and available structural information suggest that the oxytocin receptor couples to PLC through a GTP-binding protein. In support of this hypothesis, an antibody generated against the specific C-terminal region of G alpha(q) and G alpha(11) inhibits both the oxytocin-stimulated GTPase and PLC activities. This inhibition is reversed by neutralization of the antibody with the antigenic peptide. The data indicate that the oxytocin receptor couples to PLC, presumably of the beta subclass, via interaction with proteins of the G alpha(q/11) subclass. In the nonpregnant, estrogen-primed rat, the stimulation of PI turnover by oxytocin is inhibited by the hormone relaxin and by pertussis toxin. The effects of both of these agents are mediated by the action of cAMP-dependent protein kinase. In plasma membranes, GTP-stimulated PLC activity can also be inhibited by treatment with protein kinase A. These data suggest that cAMP-dependent phosphorylation at a step involving GTP-binding protein/PLC coupling can exert a negative effect on the stimulation of IP3 formation by oxytocin and thereby affect contraction/relaxation in the myometrium.
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PMID:Mechanisms regulating oxytocin receptor coupling to phospholipase C in rat and human myometrium. 871 99

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.
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PMID:Negative regulation of Raf-1 by phosphorylation of serine 621. 881 53

Previous studies have shown that the regulation of adenylyl cyclase activity is disrupted in Alzheimer's disease postmortem brain. In the present study, we determined whether disrupted adenylyl cyclase is accompanied by altered cAMP-dependent protein kinase activity in Alzheimer's disease superior temporal cortex and cerebellum. GTP gamma S-stimulated adenylyl cyclase activity was significantly lower in Alzheimer's disease superior temporal cortex, but not cerebellum, compared to values from a series of matched control cases. Neither basal or forskolin-stimulated adenylyl cyclase activities were significantly different between the Alzheimer's disease and control brain regions. No significant differences were seen in either particulate or soluble fraction cAMP-dependent protein kinase activities between the Alzheimer's disease and control brain regions. It is concluded that disrupted adenylyl cyclase signalling in Alzheimer's disease brain occurs specifically at the level of Gs-protein-enzyme interactions and is not accompanied by an altered cAMP-dependent protein kinase activity.
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PMID:Impaired G-protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain is not accompanied by reduced cyclic-AMP-dependent protein kinase A activity. 893 Mar 61

We have examined the role of cAMP-dependent protein kinase (PKA) in controlling aggregation and postaggregative development in Dictyostelium. We previously showed that cells in which the gene encoding the PKA catalytic subunit has been disrupted (pkacat- cells) are unable to aggregate [S. K. O. Mann and R. A. Firtel (1991). A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium. Mech. Dev. 35, 89-102]. We show that pkacat- cells are unable to activate adenylyl cyclase in response to cAMP stimulation due to the inability to express the aggregation-stage, G-protein-stimulated adenylyl cyclase (ACA). Constitutive expression of ACA from an actin promoter results in a high level of Mn(2+)-stimulated adenylyl cyclase activity and restores chemoattractant- and GTP gamma S-stimulated adenylyl cyclase activity but not the ability to aggregate. Similarly, expression of the constitutively active, non-G protein-coupled adenylyl cyclase ACG in pkacat- cells also does not restore the ability to aggregate, although ACG can complement cells in which the ACA gene has been disrupted. These results indicate that pkacat- cells lack multiple, essential aggregation-stage functions. As the mound forms, high, continuous levels of extracellular cAMP functioning through the cAMP serpentine receptors activate a transcriptional cascade that leads to cell-type differentiation and morphogenesis. The first step is the induction and activation of the transcription factor GBF and downstream postaggregative genes, followed by the induction of prestalk- and prespore-specific genes. We show that pkacat- cells induce postaggregative gene expression in response to exogenous cAMP, but the level of induction of some of these genes, including GBF, is reduced. SP60 (a prespore-specific gene) is not induced and ecmA (a prestalk-specific gene) is induced to very low levels. Expressing GBF constitutively in pkacat- cells restores ecmA expression to a moderate level, but SP60 is not detectably induced. Overexpression of PKAcat from the Actin 15 (Act15), ecmA prestalk, and the PKAcat promoters in pkacat- cells result in significant aberrant spatial patterning of prestalk and prespore cells, as determined by lacZ reporter studies. Our studies identify new, essential regulatory roles for PKA in mediating multicellular development.
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PMID:Role of cAMP-dependent protein kinase in controlling aggregation and postaggregative development in Dictyostelium. 912 95

The modulation of a constitutively active IRK1-like inwardly rectifying potassium channel, that is endogenously expressed in the RBL-2H3 cell, was studied with the whole-cell patch-clamp technique. Activation of G-proteins by intracellular application of GTP gamma S revealed a dual modulation of the inward rectifier. An initial increase in inward current amplitude was induced by GTP gamma S, followed by a profound inhibition of the current. The stimulation of the inward rectifier by GTP gamma S was abolished by pretreatment with pertussis toxin. The inhibitory phase of the GTP gamma S-induced response was pertussis toxin-insensitive. Stimulation of the m1-muscarinic receptor expressed in the RBL cell after stable transfection, induced an inhibition of the inwardly rectifying currents. Application of protein kinase C activators such as phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate, resulted in a strong inhibition of the currents. Application of the cAMP-dependent protein kinase activator 8-bromo cAMP also induced an inhibition of the inward rectifier. It is concluded that the inward rectifier of the RBL-2H3 cell may be inhibited both by activation of protein kinase C and by cAMP-dependent protein kinase. As this type of inward rectifier is widely expressed in the nervous system, these data imply that the channel can be inhibited by receptors that stimulate phospholipase C and/or stimulate adenylyl cyclase, and can be activated by receptors that inhibit adenylyl cyclase activity.
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PMID:Dual modulation of an inwardly rectifying potassium conductance. 914 58

1. Previous studies indicate that prostacyclin (PGI2) increases the activity of baroreceptor afferent fibres. The purpose of this study was to test the hypothesis that PGI2 inhibits Ca(2+)-activated K+ current (IK(Ca))in isolated baroreceptor neurones in culture. 2. Rat aortic baroreceptor neurones in the nodose ganglia were labelled in vivo by applying a fluorescent dye (DiI) to the aortic arch 1-2 weeks before dissociation of the neurones. Outward K+ currents in baroreceptor neurones evoked by depolarizing voltage steps from a holding potential of -40 mV were recorded using the whole-cell patch-clamp technique. 3. Exposure of baroreceptor neurones to the stable PGI2 analogue carbacyclin significantly inhibited the steady-state K+ current in a dose-dependent and reversible manner. The inhibition of K+ current was not caused indirectly by changes in cytosolic Ca2+ concentration. The Ca(2+)-activated K+ channel blocker charybdotoxin (ChTX, 10(-7) M) also inhibited the K+ current. In the presence of ChTX or in the absence of Ca2+, carbacyclin failed to inhibit the residual K+ current. Furthermore, in the presence of high concentrations of carbacyclin, ChTX did not cause further reduction of K+ current. 4. Carbacyclin-induced inhibition of IK(Ca) was mimicked by 8-bromo-cAMP and by activation of G-protein with GTP gamma S. The inhibitory effect of carbacyclin on IK(Ca) was abolished by GDP beta S, which blocks G-protein activation, and by a selective inhibitor of cAMP-dependent protein kinase, PKI5-24. 5. The results demonstrate that carbacyclin inhibits ChTX-sensitive IK(Ca) in isolated aortic baroreceptor neurones by a G-protein-coupled activation of cAMP-dependent protein kinase. This mechanism may contribute to the PGI2-induced increase in baroreceptor activity demonstrated previously.
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PMID:The prostacyclin analogue carbacyclin inhibits Ca(2+)-activated K+ current in aortic baroreceptor neurones of rats. 919

Previous studies have revealed an adenosine 3',5'-cyclic monophosphate (cAMP)-independent activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by the tyrosine kinase inhibitor genistein. To further explore its mechanism of action, we have reconstituted genistein activation of CFTR in excised inside-out membrane patches. In the presence or absence of ATP, genistein appeared unable to open silent CFTR Cl- channels. However, on CFTR prephosphorylation by cAMP-dependent protein kinase (cAK), genistein enhanced CFTR activity by twofold, resulting from a prolonged burst duration. Genistein could also hyperactivate partially phosphorylated CFTR in the absence of cAK and therefore is different from 5'-adenylylimidodiphosphate, which required fully phosphorylated CFTR. Phosphatase-resistant thiophosphorylation likewise primed the CFTR Cl- channel for hyperactivation by genistein in the absence of cAK. Replacement of ATP by GTP as a hydrolyzable nucleotide triphosphate for CFTR did not impair the ability of genistein to activate thiophosphorylated CFTR, despite the fact that GTP is a poor substrate for tyrosine kinases. These findings argue against a role of protein phosphatases or tyrosine kinases but suggest a more direct interaction of genistein with CFTR, possibly at the level of the second nucleotide-binding domain.
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PMID:Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism. 927 73

The neuropeptide oxytocin can depolarize parasympathetic preganglionic neurons in the dorsal motor nucleus of the vagus nerve of the rat by generating a sustained inward current, which is sodium-dependent and tetrodotoxin-insensitive. The second messenger activated by oxytocin receptor binding is, however, not yet known. In the present study, we attempted to characterize it by using the whole-cell recording technique and brainstem slices. When loaded with GTP-gamma-S, a non-hydrolysable analogue of GTP, vagal neurons generated a persistent inward current in the absence of agonist and the oxytocin effect was suppressed, suggesting that the peptide-evoked current was mediated by G-protein activation. Loading vagal neurons with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA) suppressed a calcium-dependent, slowly decaying potassium aftercurrent but did not affect the oxytocin response, suggesting that the latter was not mediated by an agonist-induced increase in the intracellular calcium concentration. Protein kinase C (PKC) activation was probably not involved, since the peptide-evoked current was not modified by loading neurons with the PKC inhibitor H7. Thus, the oxytocin-evoked current in vagal neurons was probably not mediated by phospholipase C-beta (PLC-beta) activation. Loading neurons with 8-Br-cAMP or with an adenylyl cyclase activator (forskolin) reduced the oxytocin-evoked current by about half. SQ 22536, an adenylyl cyclase inhibitor, reduced this current by a similar amount. However, the peptide-evoked current was unaffected by Rp-cAMPS and Sp-cAMPS, an inhibitor and an activator, respectively, of cAMP-dependent protein kinase (PKA). We suggest that oxytocin activates two distinct signalling pathways in vagal neurons: one which is cAMP-dependent, but PKA-independent, and one, unidentified, which is PLC-beta-and cAMP-independent. Each pathway accounts for about half of the peptide effect and both appear to involve G-protein activation.
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PMID:The oxytocin-induced inward current in vagal neurons of the rat is mediated by G protein activation but not by an increase in the intracellular calcium concentration. 951 66


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