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Mechanism of A-kinase-anchoring protein 79 (AKAP79) and protein kinase C interaction.

ABSTRACT: The A-kinase-anchoring protein AKAP79 co-ordinates the location of cAMP-dependent protein kinase, phosphatase 2B (PP2B/calcineurin) and protein kinase C (PKC) at postsynaptic sites in neurons. In this report we focus on the mechanism of interaction between AKAP79 and PKC. We show that neither lipid activators nor kinase activation are required for association with AKAP79. The anchoring protein binds and inhibits the conserved catalytic core of PKCbetaII. AKAP79 also associates with conventional, novel and atypical isoforms of PKC in vitro and in vivo, and immunofluorescence staining of rat hippocampal neurons demonstrates that the murine anchoring-protein homologue AKAP150 is co-distributed with PKCalpha/beta, PKCepsilon or PKCiota. Binding of the AKAP79(31-52) peptide, which inhibits kinase activity, exposes the pseudosubstrate domain of PKCbetaII, allowing endoproteinase Arg-C proteolysis in the absence of kinase activators. Reciprocal experiments have identified two arginine residues at positions 39 and 40 that are essential for AKAP79(31-52) peptide inhibition of PKCbetaII. Likewise, the same mutations in the full-length anchoring protein reduced inhibition of PKCbetaII. Thus AKAP79 associates with multiple PKC isoforms through a mechanism involving protein-protein interactions at the catalytic core where binding of the anchoring protein inhibits kinase activity through displacement of the pseudosubstrate.

SUBMITTER: Faux MC

PROVIDER: S-EPMC1220573 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Architecture and dynamics of an A-kinase anchoring protein 79 (AKAP79) signaling complex.

Project description:A-kinase anchoring protein 79 (AKAP79) is a human anchoring protein that organizes cAMP-dependent protein kinase (PKA), Ca(2+)/calmodulin (CaM)-dependent protein phosphatase (PP2B), and protein kinase C (PKC) for phosphoregulation of synaptic signaling. Quantitative biochemical analyses of selected AKAP79 complexes have determined the quaternary structure of these signaling complexes. We show that AKAP79 dimerizes, and we demonstrate that, upon addition of a lysine-reactive cross-linker, parallel homomeric dimers are stabilized through K328-K328 and K333-K333 cross-links. An assembly of greater complexity comprising AKAP79, PP2B, a type II regulatory subunit fragment (RII 1-45) of PKA, and CaM was reconstituted in vitro. Using native MS, we determined the molecular mass of this complex as 466 kDa. This indicates that dimeric AKAP79 coordinates two RII 1-45 homodimers, four PP2B heterodimers, and two CaM molecules. Binding of Ca(2+)/CaM to AKAP79 stabilizes the complex by generating a second interface for PP2B. This leads to activation of the anchored phosphatases. Our architectural model reveals how dimeric AKAP79 concentrates pockets of second messenger responsive enzyme activities at the plasma membrane.

| S-EPMC3081024 | biostudies-literature

Glycogen synthase kinase 3beta interaction protein functions as an A-kinase anchoring protein.

Project description:A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.

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Differential regulation of CaV1.2 channels by cAMP-dependent protein kinase bound to A-kinase anchoring proteins 15 and 79/150.

Project description:The CaV1.1 and CaV1.2 voltage-gated calcium channels initiate excitation-contraction coupling in skeletal and cardiac myocytes, excitation-transcription coupling in neurons, and many other cellular processes. Up-regulation of their activity by the β-adrenergic-PKA signaling pathway increases these physiological responses. PKA up-regulation of CaV1.2 activity can be reconstituted in a transfected cell system expressing CaV1.2Δ1800 truncated at the in vivo proteolytic processing site, the distal C-terminal domain (DCT; CaV1.2[1801-2122]), the auxiliary α2δ and β subunits of CaV1.2 channels, and A-kinase anchoring protein-15 (AKAP15), which binds to a site in the DCT. AKAP79/150 binds to the same site in the DCT as AKAP15. Here we report that AKAP79 is ineffective in supporting up-regulation of CaV1.2 channel activity by PKA, even though it binds to the same site in the DCT and inhibits the up-regulation of CaV1.2 channel activity supported by AKAP15. Mutation of the calcineurin-binding site in AKAP79 (AKAP79ΔPIX) allows it to support PKA-dependent up-regulation of CaV1.2 channel activity, suggesting that calcineurin bound to AKAP79 rapidly dephosphorylates CaV1.2 channels, thereby preventing their regulation by PKA. Both AKAP15 and AKAP79ΔPIX exert their regulatory effects on CaV1.2 channels in transfected cells by interaction with the modified leucine zipper motif in the DCT. Our results introduce an unexpected mode of differential regulation by AKAPs, in which binding of different AKAPs at a single site can competitively confer differential regulatory effects on the target protein by their association with different signaling proteins.

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High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides.

Project description:PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.

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A-Kinase Anchoring Protein 79/150 Scaffolds Transient Receptor Potential A 1 Phosphorylation and Sensitization by Metabotropic Glutamate Receptor Activation.

Project description:Mechanical pain serves as a base clinical symptom for many of the world's most debilitating syndromes. Ion channels expressed by peripheral sensory neurons largely contribute to mechanical hypersensitivity. Transient Receptor Potential A 1 (TRPA1) is a ligand-gated ion channel that contributes to inflammatory mechanical hypersensitivity, yet little is known as to the post-translational mechanism behind its somatosensitization. Here, we utilize biochemical, electrophysiological, and behavioral measures to demonstrate that metabotropic glutamate receptor-induced sensitization of TRPA1 nociceptors stimulates targeted modification of the receptor. Type 1 mGluR5 activation increases TRPA1 receptor agonist sensitivity in an AKA-dependent manner. As a scaffolding protein for Protein Kinases A and C (PKA and PKC, respectively), AKAP facilitates phosphorylation and sensitization of TRPA1 in ex vivo sensory neuronal preparations. Furthermore, hyperalgesic priming of mechanical hypersensitivity requires both TRPA1 and AKAP. Collectively, these results identify a novel AKAP-mediated biochemical mechanism that increases TRPA1 sensitivity in peripheral sensory neurons, and likely contributes to persistent mechanical hypersensitivity.

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The A-Kinase Anchoring Protein (AKAP) Glycogen Synthase Kinase 3β Interaction Protein (GSKIP) Regulates β-Catenin through Its Interactions with Both Protein Kinase A (PKA) and GSK3β.

Project description:The A-kinase anchoring protein (AKAP) GSK3β interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3β are required for the regulation of β-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets β-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause β-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the β-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3β facilitates control of the destabilizing phosphorylation of β-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on β-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with β-catenin. The regulation of β-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3β, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3β is a conserved GSK3β interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3β by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.

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Bioinformatic design of A-kinase anchoring protein-in silico: a potent and selective peptide antagonist of type II protein kinase A anchoring.

Project description:Compartmentalization of the cAMP-dependent protein kinase (PKA) is coordinated through association with A-kinase anchoring proteins (AKAPs). A defining characteristic of most AKAPs is a 14- to 18-aa sequence that binds to the regulatory subunits (RI or RII) of the kinase. Cellular delivery of peptides to these regions disrupts PKA anchoring and has been used to delineate a physiological role for AKAPs in the facilitation of certain cAMP-responsive events. Here, we describe a bioinformatic approach that yields an RII-selective peptide, called AKAP-in silico (AKAP-IS), that binds RII with a K(d) of 0.4 nM and binds RI with a K(d) of 277 nM. AKAP-IS associates with the type II PKA holoenzyme inside cells and displaces the kinase from natural anchoring sites. Electrophysiological recordings indicate that perfusion of AKAP-IS evokes a more rapid and complete attenuation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents than previously described anchoring inhibitor peptides. Thus, computer-based and peptide array screening approaches have generated a reagent that binds PKA with higher affinity than previously described AKAPs.

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A-kinase anchoring protein targeting of protein kinase A and regulation of HERG channels.

Project description:Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K+ current (I(Kr)), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I(Kr) by cAMP.

| S-EPMC2522378 | biostudies-literature

Cypher/ZASP is a novel A-kinase anchoring protein.

Project description:PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser(265) and Ser(296) by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser(1928) induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.

| S-EPMC3795241 | biostudies-literature

Protein Kinase A Regulates Platelet Phosphodiesterase 3A through an A-Kinase Anchoring Protein Dependent Manner.

Project description:Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI2)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.

| S-EPMC11240354 | biostudies-literature

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