Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2.
ABSTRACT: Generation of the second messenger molecule cAMP mediates a variety of cellular responses which are essential for critical cellular processes. In response to elevated cAMP levels, cAMP dependent protein kinase (PKA) phosphorylates serine and threonine residues on a wide variety of target substrates. In order to enhance the precision and directionality of these signaling events, PKA is localized to discrete locations within the cell by A-kinase anchoring proteins (AKAPs). The interaction between PKA and AKAPs is mediated via an amphipathic ?-helix derived from AKAPs which binds to a stable hydrophobic groove formed in the dimerization/docking (D/D) domain of PKA-R in an isoform-specific fashion. Although numerous AKAP disruptors have previously been identified that can inhibit either RI- or RII-selective AKAPs, no AKAP disruptors have been identified that have isoform specificity for RI? versus RI? or RII? versus RII?. As a strategy to identify isoform-specific AKAP inhibitors, a library of chemically stapled protein-protein interaction (PPI) disruptors was developed based on the RII-selective AKAP disruptor, STAD-2. An alanine was substituted at each position in the sequence, and from this library it was possible to delineate the importance of longer aliphatic residues in the formation of a region which complements the hydrophobic cleft formed by the D/D domain. Interestingly, lysine residues that were added to both terminal ends of the peptide sequence to facilitate water solubility appear to contribute to isoform specificity for RII? over RII? while having only weak interaction with RI. This work supports current hypotheses on the mechanisms of AKAP binding and highlights the significance of particular residue positions that aid in distinguishing between the RII isoforms and may provide insight into future design of isoform-selective AKAP disruptors.
Project description:A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RI?, but not RII?, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.
Project description:A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells.
Project description:Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein-protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIalpha isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4+/-0.2 nM and 6+/-1 nM respectively). In contrast, RIalpha isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156+/-10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13+/-1 nM) was 20-fold more potent than PV-38 (IC50 of 304+/-17 nM) and did not compete in the RIIalpha-AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA-AKAP complexes and competition of RII-AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIalpha-AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA-AKAP complexes.
Project description:Protein kinase A-anchoring proteins (AKAPs) provide spatio-temporal specificity for the omnipotent cAMP-dependent protein kinase (PKA) via high affinity interactions with PKA regulatory subunits (PKA-RI, RII). Many PKA-RII-AKAP complexes are heavily tethered to cellular substructures, whereas PKA-RI-AKAP complexes have remained largely undiscovered. Here, using a cAMP affinity-based chemical proteomics strategy in human heart and platelets, we uncovered a novel, ubiquitously expressed AKAP, termed small membrane (sm)AKAP due to its specific localization at the plasma membrane via potential myristoylation/palmitoylation anchors. In vitro binding studies revealed specificity of smAKAP for PKA-RI (K(d) = 7 nM) over PKA-RII (K(d) = 53 nM) subunits, co-expression of smAKAP with the four PKA R subunits revealed an even more exclusive specificity of smAKAP for PKA-RI?/? in the cellular context. Applying the singlet oxygen-generating electron microscopy probe miniSOG indicated that smAKAP is tethered to the plasma membrane and is particularly dense at cell-cell junctions and within filopodia. Our preliminary functional characterization of smAKAP provides evidence that, like PKA-RII, PKA-RI can be tightly tethered by a novel repertoire of AKAPs, providing a new perspective on spatio-temporal control of cAMP signaling.
Project description:A-Kinase anchoring proteins (AKAPs) act as spatial and temporal regulators of protein kinase?A (PKA) by localizing PKA along with multiple proteins into discrete signaling complexes. AKAPs interact with the PKA holoenzyme through an ?-helix that docks into a groove formed on the dimerization/docking domain of PKA-R in an isoform-dependent fashion. In an effort to understand isoform selectivity at the molecular level, a library of protein-protein interaction (PPI) disruptors was designed to systematically probe the significance of an aromatic residue on the AKAP docking sequence for RI selectivity. The stapled peptide library was designed based on a high affinity, RI-selective disruptor of AKAP binding, RI-STAD-2. Phe, Trp and Leu were all found to maintain RI selectivity, whereas multiple intermediate-sized hydrophobic substitutions at this position either resulted in loss of isoform selectivity (Ile) or a reversal of selectivity (Val). As a limited number of RI-selective sequences are currently known, this study aids in our understanding of isoform selectivity and establishing parameters for discovering additional RI-selective AKAPs.
Project description:A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18? bound to the D/D domain of the regulatory RII? subunits of PKA. We identified three hydrophilic anchor points in AKAP18? outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions.
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.
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.
Project description:cAMP regulates cellular functions primarily by activating PKA. The involvement of PKAs in various signaling pathways occurring simultaneously in different cellular compartments necessitates stringent spatial and temporal regulation. This specificity is largely achieved by binding of PKA to protein scaffolds, whereby a distinct group of proteins called A kinase anchoring proteins (AKAPs) play a dominant role. AKAPs are a diverse family of proteins that all bind via a small PKA binding domain to the regulatory subunits of PKA. The binding affinities between PKA and several AKAPs can be different for different isoforms of the regulatory subunits of PKA. Here we employ a combination of affinity chromatography and mass spectrometry-based quantitative proteomics to investigate specificity in PKA-AKAP interactions. Three different immobilized cAMP analogs were used to enrich for PKA and its interacting proteins from several systems; HEK293 and RCC10 cells and rat lung and testis tissues. Stable isotope labeling was used to confidently identify and differentially quantify target proteins and their preferential binding affinity for the three different cAMP analogs. We were able to enrich all four isoforms of the regulatory subunits of PKA and concomitantly identify more than 10 AKAPs. A selective enrichment of the PKA RI isoforms could be achieved; which allowed us to unravel which AKAPs bind preferentially to the RI or RII regulatory domains of PKA. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual specificity with a potential preference for RI. For some of these AKAPs our data provide the first insights into their specificity.
Project description:Toll-like receptors (TLRs) direct a proinflammatory program in macrophages. One mediator whose generation is induced by TLR ligation is prostaglandin E(2) (PGE(2)), which is well known to increase intracellular cAMP upon G protein-coupled receptor ligation. How PGE(2)/cAMP shapes the nascent TLR response and the mechanisms by which it acts remain poorly understood. Here we explored PGE(2)/cAMP regulation of NO production in primary rat alveolar macrophages stimulated with the TLR4 ligand LPS. Endogenous PGE(2) synthesis accounted for nearly half of the increment in NO production in response to LPS. The enhancing effect of PGE(2) on LPS-stimulated NO was mediated via cAMP, generated mainly upon ligation of the E prostanoid 2 receptor and acting via protein kinase A (PKA) rather than via the exchange protein activated by cAMP. Isoenzyme-selective cAMP agonists and peptide disruptors of protein kinase A anchoring proteins (AKAPs) implicated PKA regulatory subunit type I (RI) interacting with an AKAP in this process. Gene knockdown of potential RI-interacting AKAPs expressed in alveolar macrophages revealed that AKAP10 was required for PGE(2) potentiation of LPS-induced NO synthesis. AKAP10 also mediated PGE(2) potentiation of the expression of cytokines IL-10 and IL-6, whereas PGE(2) suppression of TNF-? was mediated by AKAP8-anchored PKA-RII. Our data illustrate the pleiotropic manner in which G protein-coupled receptor-derived cAMP signaling can influence TLR responses in primary macrophages and suggest that AKAP10 may coordinate increases in gene expression.