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

Compartmentalization of the type II cAMP-dependent protein kinase is conferred by interaction of the regulatory subunit (RII) with A-Kinase Anchoring Proteins (AKAPs). The AKAP-binding site involves amino-terminal residues on each RII protomer and is formed through dimerization. A site-directed mutagenesis strategy was utilized to assess the contribution of individual residues in either RII isoform, RIIalpha or RIIbeta, for interaction with various anchoring proteins. Substitution of long-chain or bulky hydrophobic groups (leucines or phenylalanines) for isoleucines at positions 3 and 5 in RIIalpha decreased AKAP-binding up to 24 +/- 3 (n = 8)-fold, whereas introduction of valines had minimal effects. Replacement with hydrophilic residues (serine or asparigine) at both positions abolished AKAP binding. Mutation of proline 6 in RIIalpha reduced binding for four AKAPs (Ht31, MAP2, AKAP79, and AKAP95) from 2.3 to 20-fold (n = 4) whereas introduction of an additional proline at position 6 in RIIbeta increased or conferred binding toward these anchoring proteins. Therefore, we conclude that beta-branched side chains at positions 3 and 5 are favored determinants for AKAP-binding and prolines at positions 6 and 7 increase or stabilize RIIalpha interaction with selected anchoring proteins.
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PMID:Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII. Classification Of side chain determinants for anchoring and isoform selective association with AKAPs. 891 May 53

Activation of D1-like dopamine (DA) receptors reduces peak Na(+) current in acutely isolated hippocampal neurons via a modulatory mechanism involving phosphorylation of the Na(+) channel alpha subunit by cAMP-dependent protein kinase (PKA). Peak Na(+) current is reduced 20-50% in the presence of the D1 agonist SKF 81297 or the PKA activator Sp-5,6-dichloro-l-beta-d-ribofuranosyl benzimidazole-3',5'-cyclic monophosphorothionate (cBIMPS). Co-immunoprecipitation experiments show that Na(+) channels are associated with PKA and A-kinase-anchoring protein 15 (AKAP-15), and immunocytochemical labeling reveals their co-localization in the cell bodies and proximal dendrites of hippocampal pyramidal neurons. Anchoring of PKA near the channel by an AKAP, which binds the RII alpha regulatory subunit, is necessary for Na(+) channel modulation in acutely dissociated hippocampal pyramidal neurons. Intracellular dialysis with the anchoring inhibitor peptides Ht31 from a human thyroid AKAP and AP2 from AKAP-15 eliminated the modulation of the Na(+) channel by the D1-agonist SKF 81297 and the PKA activator cBIMPS. In contrast, dialysis with the inactive proline-substituted control peptides Ht31-P and AP2-P had little effect on the D1 and PKA modulation. Therefore, we conclude that modulation of the Na(+) channel by activation of D1-like DA receptors requires targeted localization of PKA near the channel to achieve phosphorylation of the alpha subunit and to modify the functional properties of the channel.
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PMID:Dopaminergic modulation of voltage-gated Na+ current in rat hippocampal neurons requires anchoring of cAMP-dependent protein kinase. 1046 Feb 75

A-kinase anchoring proteins tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of PKA (RII) and the RII-binding domain of the human thyroid RII anchoring protein (Ht31), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to enhanced yellow fluorescent protein (EYFP), Ht31 was linked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, Ht31.ECFP, an increase in emission of the acceptor fluorophore, RII.EYFP, and a decrease in emission from Ht31.ECFP were observed. The emission ratio (acceptor/donor) was increased 2-fold (p < 0.05) in cells expressing Ht31.ECFP and RII.EYFP compared with cells expressing Ht31P.ECFP, the inactive form of Ht31, and RII.EYFP. These results provide the first in vivo demonstration of RII/Ht31 interaction in living cells and confirm previous in vitro findings of RII/Ht31 binding. Using surface plasmon resonance, we also showed that the green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the PKA signaling pathway in living cells.
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PMID:Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins. 1055 79

During pregnancy in the rat, there is a change in the ability of chlorophenylthio (CPT)-cAMP to inhibit myometrial phosphatidylinositide turnover. This is accompanied by a change in the association of proteins with a plasma membrane A kinase anchoring protein (AKAP). Both CPT-cAMP and isoproterenol inhibited oxytocin-stimulated phosphatidylinositide turnover on days 12 through 20 of gestation, whereas neither agent had an effect on day 21. Accompanying this change was a dramatic decrease in the concentration and activity of cAMP-dependent protein kinase [protein kinase A (PKA)] and an increase in the concentration of protein phosphatase 2B (PP2B) in plasma membranes from day 21 compared with day 19 pregnant rats. In contrast, both PKA and PP2B concentrations and activities increased in total myometrial homogenates. Both PKA and PP2B coimmunoprecipitated with an antibody against the 150-kDa AKAP found in rat myometrial plasma membranes. More PKA was associated with AKAP150 on day 19 than on day 21, while the reverse was true for PP2B. Disruption of PKA/AKAP association in day 19 pregnant rat myometrial cells with the specific interaction inhibitor peptide S-Ht31 resulted in the loss of the cAMP-inhibitory effect on phosphatidylinositide turnover. PP2B activity in myometrial homogenates dephosphorylated PLCbeta3, a PKA substrate targeted in the inhibition of Galphaq-stimulated phosphatidylinositide turnover. The dramatic loss of the cAMP-inhibitory effect on day 21 of pregnancy may alter the balance between uterine contraction and relaxation near parturition. The changes in the relative concentrations of PKA and PP2B associated with AKAP150 are consistent with a functional role for AKAP150 scaffolding in the alteration of cellular signaling.
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PMID:A role for AKAP (A kinase anchoring protein) scaffolding in the loss of a cyclic adenosine 3',5'-monophosphate inhibitory response in late pregnant rat myometrium. 1059 75

Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.
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PMID:Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding. 1076 1

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.
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PMID:Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin. 1079 17

Downstream regulation of the cAMP-dependent protein kinase (PKA) pathway is mediated by anchoring proteins (AKAPs) that sequester PKA to specific subcellular locations through binding to PKA regulatory subunits (RI or RII). The RII-binding domain of all AKAPs forms an amphipathic alpha-helix with similar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms that regulate AKAP-PKA affinity are not clearly defined. Using surface plasmon resonance (SPR) spectroscopy, we measured real-time kinetics of RII interaction with various AKAPs. Base-line equilibrium binding constants (K(d)) for RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nm, 119 nm, and 6.6 microm, respectively. PKA stimulation of intact Chinese hamster ovary cells increased RIIalpha binding to AKAP100/mAKAP and AKAP15/18 by approximately 7- and 82-fold, respectively. These results suggest that differences in primary sequence of the RII-binding domain may be responsible for the selective affinity of RII for different AKAPs. Furthermore, RII autophosphorylation may provide additional localized regulation of kinase anchoring. In cardiac myocytes, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be involved in adding additional specificity in intracellular signaling in diverse cell types and under conditions of cAMP/PKA activation.
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PMID:Selectivity and regulation of A-kinase anchoring proteins in the heart. The role of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase. 1099 82

The specificity of intracellular signaling events is controlled, in part, by compartmentalization of protein kinases and phosphatases. The subcellular localization of these enzymes is often maintained by protein- protein interactions. A prototypic example is the compartmentalization of the cAMP-dependent protein kinase (PKA) through its association with A-kinase anchoring proteins (AKAPs). A docking and dimerization domain (D/D) located within the first 45 residues of each regulatory (R) subunit protomer forms a high affinity binding site for its anchoring partner. We now report the structures of two D/D-AKAP peptide complexes obtained by solution NMR methods, one with Ht31(493-515) and the other with AKAP79(392-413). We present the first direct structural data demonstrating the helical nature of the peptides. The structures reveal conserved hydrophobic interaction surfaces on the helical AKAP peptides and the PKA R subunit, which are responsible for mediating the high affinity association in the complexes. In a departure from the dimer-dimer interactions seen in other X-type four-helix bundle dimeric proteins, our structures reveal a novel hydrophobic groove that accommodates one AKAP per RIIalpha D/D.
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PMID:A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes. 1128 29

A possible mechanism(s) behind exercise training-enhanced lipolysis was investigated in rat adipocytes. Exercise training (9 weeks; running) enhanced the activity of cAMP-dependent protein kinase (PKA) and the protein expressions of PKA subunits (catalytic, RII alpha, and RII beta) in P(40) fraction (sedimenting at 40,000g), but not in I(40) fraction (infranatant of 40,000g) of adipocyte homogenate. The expression of PKA-anchoring protein 150 (AKAP150) in P(40) fraction was greater in exercise-trained (TR) than in control (C) rats. Hormone-sensitive lipase (HSL) activities in both fractions were also greater in TR. On the other hand, stimulated lipolysis was accompanied by increased activities of HSL in P(40) but not in I(40) fraction. The decreases in stimulated lipolysis due to St-Ht31 were greater in TR rats. Thus, the mechanisms behind exercise training-enhanced adipocyte lipolysis could involve the increased activities of PKA and HSL with enhanced expressions of AKAP150 and some subunits of PKA, all of which may be compartmentalized within adipocytes.
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PMID:Possible mechanisms by which adipocyte lipolysis is enhanced in exercise-trained rats. 1215 Sep 37

Activation of the cAMP-dependent protein kinase (PKA) is critical for both short- and long-term facilitation in Aplysia sensory neurons. There are two types of the kinase, I and II, differing in their regulatory (R) subunits. We cloned Aplysia RII; RI was cloned previously. Type I PKA is mostly soluble in the cell body whereas type II is enriched at nerve endings where it is bound to two prominent A kinase-anchoring-proteins (AKAPs). Disruption of the binding of RII to AKAPs by Ht31, an inhibitory peptide derived from a human thyroid AKAP, prevents both the short- and the long-term facilitation produced by serotonin (5-HT). During long-term facilitation, RII is transcriptionally upregulated; in contrast, the amount of RI subunits decreases, and previous studies have indicated that the decrease is through ubiquitin-proteosome-mediated proteolysis. Experiments with antisense oligonucleotides injected into the sensory neuron cell body show that the increase in RII protein is essential for the production of long-term facilitation. Using synaptosomes, we found that 5-HT treatment causes RII protein to increase at nerve endings. In addition, using reverse transcription-PCR, we found that RII mRNA is transported from the cell body to nerve terminals. Our results suggest that type I operates in the nucleus to maintain cAMP response element-binding protein-dependent gene expression, and type II PKA acts at sensory neuron synapses phosphorylating proteins to enhance release of neurotransmitter. Thus, the two types of the kinase have distinct but complementary functions in the production of facilitation at synapses of an identified neuron.
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PMID:The two regulatory subunits of aplysia cAMP-dependent protein kinase mediate distinct functions in producing synaptic plasticity. 1501 22


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