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: 4.6). 04/2013; 288(24). DOI: 10.1074/jbc.M112.447326
Source: PubMed

ABSTRACT 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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Available from: Matthew G Gold, Jul 07, 2015
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