The inhibitory effect of cyclic AMP on phosphatidylinositol kinase is not mediated by the cAMP dependent protein kinase

In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.

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Ca2+ and lipolysis in adipocytes from exercise-trained rats

Journal of Applied Physiology ◽

10.1152/jappl.1994.77.6.2618 ◽

1994 ◽

Vol 77 (6) ◽

pp. 2618-2624 ◽

Cited By ~ 12

Author(s):

T. Izawa ◽

T. Komabayashi

Keyword(s):

Protein Kinase ◽

Adrenocorticotropic Hormone ◽

Kinase Activity ◽

Inhibitory Effect ◽

Protein Kinase Activity ◽

Dibutyryl Camp ◽

Inhibitory Effects ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

The effects of Ca2+ on lipolysis and protein kinase activity in adipocytes from exercise-trained rats were investigated. Chronic exercise significantly increased lipolytic responses to norepinephrine and dibutyryl adenosine 3′,5′-cyclic monophosphate (cAMP). The inhibitory effects of N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a calumodulin inhibitor, on norepinephrine- and dibutyryl cAMP-stimulated lipolysis were significantly greater in trained than in sedentary rats. Training did not alter cAMP-dependent protein kinase activity. However, the inhibitory effect of W-7 on cAMP-dependent protein kinase activity was much greater in trained than in sedentary rats. The basal intracellular free Ca2+ concentration ([Ca2+]i) was significantly higher in trained than in sedentary rats. The rapid and transient increases in [Ca2+]i due to adrenocorticotropic hormone and phenylephrine from basal levels were significantly lower in trained than in sedentary rats. However, the higher basal [Ca2+]i level in trained rats led to increases in sustained [Ca2+]i levels after stimulation. We concluded that in trained rats the regulation of protein kinase activity by cAMP depends to a greater degree on Ca(2+)-calmodulin complex than it does in sedentary rats and that training alters adipocyte intracellular Ca2+ homeostasis, including [Ca2+]i responsiveness to hormones.

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Involvement of cAMP-Dependent Protein Kinase in μ-Opioid Modulation of NMDA-Mediated Synaptic Currents

Journal of Neurophysiology ◽

10.1152/jn.1997.78.2.759 ◽

1997 ◽

Vol 78 (2) ◽

pp. 759-766 ◽

Cited By ~ 23

Author(s):

Cui-Wei Xie ◽

Darrell V. Lewis

Keyword(s):

Nmda Receptor ◽

Protein Kinase ◽

G Proteins ◽

Inhibitory Action ◽

Granule Cells ◽

Inhibitory Effect ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Dependent Protein ◽

Camp Cascade

Xie, Cui-Wei and Darrell V. Lewis. Involvement of cAMP-dependent protein kinase in μ-opioid modulation of NMDA-mediated synaptic currents. J. Neurophysiol. 78: 759–766, 1997. We have previously reported dual effects of μ-opioids on N-methyl-d-aspartate (NMDA)-receptor-mediated synaptic events in the hippocampal dentate gyrus: an indirect facilitating effect via suppression of GABAergic interneurons (disinhibition) and a direct inhibitory effect in the presence of γ-aminobutyric acid-A (GABAA) antagonists. The cellular mechanism underlying the inhibitory effect of μ-opioids remains to be determined. In the present study we examine the role of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in μ-opioid-induced inhibition of NMDA currents in rat hippocampal slices. NMDA-receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were evoked by stimulating the lateral perforant path and were recorded from dentate granule cells with the use of whole cell voltage-clamp techniques in the presence of the GABAA antagonist and a non-NMDA type of glutamate receptor antagonist. Two selective μ-agonists, [N-MePhe3, D-Pro4]-morphiceptin and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin, induced dose-dependent inhibition of NMDA EPSCs in a concentration range of 0.3–10 μM. This inhibitory effect could be completely reversed by the opioid antagonists naloxone or prevented by a selective μ-antagonist cyprodime, but was not affected by removal of Mg2+ from the external perfusion medium. Intracellular application of pertussis toxin (PTX) into the granule cell via whole cell recording pipettes completely prevented μ-opioid-induced reduction in NMDA currents, suggesting that a postsynaptic mechanism involving PTX-sensitive G proteins might be responsible for the inhibitory action of μ-opioids. Further studies were conducted to identify the intracellular messengers that coupled with G proteins and transduced the effect of μ-opioids in granule cells. The adenylate cyclase activator forskolin was found to enhance NMDA-receptor-mediated synaptic responses and to reverse the inhibitory effect of μ-opioids. Sp-cAMPS, a specific PKA activator, also enhanced NMDA EPSCs, whereas the PKA inhibitor Rp-cAMPS reduced NMDA EPSCs and occluded further inhibition of the current by μ-opioids. These findings strongly suggest that NMDA receptor function is subject to the modulation by PKA, and that μ-opioids can inhibit NMDA currents through suppression of the cAMP cascade in the postsynaptic neuron. Combined with our previous findings, the present results also indicate that μ-opioids can modulate NMDA-receptor-mediated synaptic activity in a complex manner. The net effect of μ-opioids in the dentate gyrus may depend on the interplay between its disinhibitory action, which facilitates NMDA-receptor-mediated responses, and its inhibitory action on the cAMP cascade.

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Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. II. Temporal and spatial kinetics.

The Journal of Cell Biology ◽

10.1083/jcb.93.3.727 ◽

1982 ◽

Vol 93 (3) ◽

pp. 727-734 ◽

Cited By ~ 29

Author(s):

C V Byus ◽

W H Fletcher

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Activity Ratio ◽

Hepatoma Cells ◽

Catalytic Subunit ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Dependent Protein ◽

Temporal And Spatial

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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Faculty Opinions recommendation of Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1115508.571542 ◽

2008 ◽

Author(s):

Silvana Curci ◽

Andrea Gerbino

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Adipocyte Differentiation ◽

Synergistic Action ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Dependent Protein ◽

Dependent Processes ◽

Stimulation Of

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

Biochemical Journal ◽

10.1042/bj2510757 ◽

1988 ◽

Vol 251 (3) ◽

pp. 757-762 ◽

Cited By ~ 131

Author(s):

J D Rothermel ◽

L H Parker Botelho

Keyword(s):

Protein Kinase ◽

Linear Regression ◽

Cyclic Amp ◽

Competitive Inhibitor ◽

Type Ii ◽

Sulphur Atom ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Activation Data ◽

Dependent Protein

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.

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Interaction of acetylcholine and epinephrine on heart cyclic AMP-dependent protein kinase

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1978.234.4.h432 ◽

1978 ◽

Vol 234 (4) ◽

pp. H432-H438

Author(s):

S. L. Keely ◽

T. M. Lincoln ◽

J. D. Corbin

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Inhibitory Effect ◽

Contractile Force ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Kinase Activation ◽

Camp Dependent Protein Kinase ◽

Cholinergic Agent ◽

Dependent Protein

In the isolated perfused rat heart, epinephrine produced a rapid, concentration-dependent increase in cyclic adenosine 3',5'-monophosphate (cAMP), activation of cAMP-dependent protein kinase, activation of phosphorylase, and increase in contractile force. At epinephrine concentrations of 1 micron or less, acetylcholine antagonized all these beta-adrenergic effects and also increased cyclic guanosine 3',5'-monophosphate (cGMP) levels. When used alone, acetylcholine produced a rapid elevation of cGMP and markedly diminished contractile force but did not significantly lower basal cAMP levels or cAMP-dependent protein kinase activity. The data suggest that changes in cAMP-dependent protein kinase activity can explain the antagonism of epinephrine-induced activation of phosphorylase by acetylcholine, but cannot completely account for the inhibitory effect of the cholinergic agent on contractile force.

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