Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection

University College London, United Kingdom
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2013; 288(24). DOI: 10.1074/jbc.M112.447326
Source: PubMed


PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with
A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved
AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII
mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments
validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the
contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed
the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this
structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.

Download full-text


Available from: Matthew G Gold
  • Source
    • "hich used immobilized AKAP helices to enrich for phage variants that displayed mutant RII D / D domains , selected variants that exhibit preferential binding to specific AKAPs . These mutant D / D domains are termed Rselects , and have been shown in preliminary work to bind and label AKAPs in a cellular context as well as in to purified proteins ( Gold et al . , 2013 ) . Further development of these Rselects could lead to high affinity binding variants that could disrupt individual pools of anchored PKA while allowing other anchored PKA signaling events to proceed unperturbed . The potential to isolate spatially constrained post - translational modifications is an important step forward for targeted"
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphorylation events that occur in response to the second messenger cAMP are controlled spatially and temporally by protein kinase A (PKA) interacting with A-kinase anchoring proteins (AKAPs). Recent advances in understanding the structural basis for this interaction have reinforced the hypothesis that AKAPs create spatially constrained signaling microdomains. This has led to the realization that the PKA/AKAP interface is a potential drug target for modulating a plethora of cell-signaling events. Pharmacological disruption of kinase-AKAP interactions has previously been explored for disease treatment and remains an interesting area of research. However, disrupting or enhancing the association of phosphatases with AKAPs is a therapeutic concept of equal promise, particularly since they oppose the actions of many anchored kinases. Accordingly, numerous AKAPs bind phosphatases such as protein phosphatase 1 (PP1), calcineurin (PP2B), and PP2A. These multimodal signaling hubs are equally able to control the addition of phosphate groups onto target substrates, as well as the removal of these phosphate groups. In this review, we describe recent advances in structural analysis of kinase and phosphatase interactions with AKAPs, and suggest future possibilities for targeting these interactions for therapeutic benefit.
    Full-text · Article · Aug 2015 · Frontiers in Pharmacology
  • Source
    • "Phopshorylated forms of Drp1 cannot promotes mitochondrial fission therefore high PKA activity at the outer mitochondrial membrane blocks mitochondrial fission resulting in elongated organelles. and cardiovascular disease, among others (Zakhary et al., 2000; Chen et al., 2007; Wirtenberger et al., 2007; Gold et al., 2013b). As our understanding of the mechanisms that underlie compartmentalized signaling builds up, ideas are also developing about how to selectively manipulate this signaling pathway locally for therapeutic purposes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In the complex microcosm of a cell, information security and its faithful transmission are critical for maintaining internal stability. To achieve a coordinated response of all its parts to any stimulus the cell must protect the information received from potentially confounding signals. Physical segregation of the information transmission chain ensures that only the entities able to perform the encoded task have access to the relevant information. The cAMP intracellular signaling pathway is an important system for signal transmission responsible for the ancestral 'flight or fight' response and involved in the control of critical functions including frequency and strength of heart contraction, energy metabolism and gene transcription. It is becoming increasingly apparent that the cAMP signaling pathway uses compartmentalization as a strategy for coordinating the large number of key cellular functions under its control. Spatial confinement allows the formation of cAMP signaling "hot spots" at discrete subcellular domains in response to specific stimuli, bringing the information in proximity to the relevant effectors and their recipients, thus achieving specificity of action. In this report we discuss how the different constituents of the cAMP pathway are targeted and participate in the formation of cAMP compartmentalized signaling events. We illustrate a few examples of localized cAMP signaling, with a particular focus on the nucleus, the sarcoplasmic reticulum and the mitochondria. Finally, we discuss the therapeutic potential of interventions designed to perturb specific cAMP cascades locally.
    Full-text · Article · Apr 2014 · Pharmacology [?] Therapeutics
  • Source
    • "Several structural studies have established that anchoring helices of AKAPs dock into a shallow hydrophobic groove that is formed by the dimerization of the D/D domains of RI (Sarma et al., 2010) or RII (Gold et al., 2006; Kinderman et al., 2006). In addition, highthroughput sequence–function approaches (Fowler et al., 2010) have confirmed a role for hydrophilic interaction sites on the D/D surface (Gold et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The second messenger cyclic AMP (cAMP) operates in discrete subcellular regions within which proteins that synthesize, break down or respond to the second messenger are precisely organized. A burgeoning knowledge of compartmentalized cAMP signaling is revealing how the local control of signaling enzyme activity impacts upon disease. The aim of this Cell Science at a Glance article and the accompanying poster is to highlight how misregulation of local cyclic AMP signaling can have pathophysiological consequences. We first introduce the core molecular machinery for cAMP signaling, which includes the cAMP-dependent protein kinase (PKA), and then consider the role of A-kinase anchoring proteins (AKAPs) in coordinating different cAMP-responsive proteins. The latter sections illustrate the emerging role of local cAMP signaling in four disease areas: cataracts, cancer, diabetes and cardiovascular diseases.
    Full-text · Article · Oct 2013 · Journal of Cell Science
Show more