Engineering A-kinase anchoring protein (AKAP)-selective regulatory subunits of protein kinase A (PKA) through structure-based phage selection

J Biol Chem. 2013 Jun 14;288(24):17111-21. doi: 10.1074/jbc.M112.447326. Epub 2013 Apr 26.


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.

Keywords: AKAP; Cell Biology; Compartmentalization; Peptide Arrays; Phage Display; Protein Kinase A (PKA); Structure-based Design.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • A Kinase Anchor Proteins / chemistry*
  • A Kinase Anchor Proteins / genetics
  • A Kinase Anchor Proteins / metabolism
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Cell Surface Display Techniques*
  • Consensus Sequence
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Structure, Secondary
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Protein Transport
  • Sequence Analysis, DNA


  • A Kinase Anchor Proteins
  • AKAP2 protein, human
  • AKAP5 protein, human
  • AKAP7 protein, human
  • Membrane Proteins
  • Peptide Fragments
  • Protein Subunits