title: Engineering A-kinase anchoring protein (AKAP)-selective regulatory subunits of protein kinase A (PKA) through structure-based phage selection.
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.
authors.affiliation: Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195, USA. m.gold@ucl.ac.uk
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Gold
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Fowler
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Means
authors.forename: Catherine T
authors.initials: CT
authors.lastname: Pawson
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Stephany
authors.forename: Lorene K
authors.initials: LK
authors.lastname: Langeberg
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
authors.forename: John D
authors.initials: JD
authors.lastname: Scott
mesh_terms: D054758:A Kinase Anchor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D061505:Cell Surface Display Techniques; D016384:Consensus Sequence; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D017422:Sequence Analysis, DNA
chemical_list: D054758:A Kinase Anchor Proteins; C484688:AKAP2 protein, human; C487775:AKAP5 protein, human; C112596:AKAP7 protein, human; D008565:Membrane Proteins; D010446:Peptide Fragments; D021122:Protein Subunits
keywords: AKAP; Cell Biology; Compartmentalization; Peptide Arrays; Phage Display; Protein Kinase A (PKA); Structure-based Design