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


The cAMP-dependent protein kinase (PKA) is retained within distinct subcellular environments by the association of its type-II (RII) regulatory 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 structural-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.

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    • "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"
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    • "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. "
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    • "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). "
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