Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection |
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Authors: | Matthew G. Gold Douglas M. Fowler Christopher K. Means Catherine T. Pawson Jason J. Stephany Lorene K. Langeberg Stanley Fields John D. Scott |
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Affiliation: | From the ‡Howard Hughes Medical Institute and ;the Departments of §Pharmacology.;¶Genome Sciences, and ;‖Medicine, University of Washington School of Medicine, Seattle, Washington 98195 |
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Abstract: | 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. |
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Keywords: | AKAP Cell Biology Peptide Arrays Phage Display Protein Kinase A (PKA) Compartmentalization Structure-based Design |
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