Selection and Characterization of Ligand‐Regulated Peptides that Inhibit the 5′AMP‐Activated Protein Kinase

We have developed a system for the selection of Ligand‐Regulated Peptides (LiRPs), whereby libraries of peptides can be displayed from an FKBP‐FRB‐GST protein scaffold and their interaction with a target protein can be modulated with the small molecule Rapamycin. We have used our LiRP scaffold system to select peptides for their ability to bind to the 5′AMP‐Activated Protein Kinase (AMPK) á1 subunit in a yeast two‐hybrid format. Isolated LiRPs shared a high sequence similarity and were of the general sequence BR(Q, M) RXXX. These LiRPs were shown to interact with both the AMPK á1 and á2 catalytic subunits n‐terminal kinase domain region (amino acids 1–270). These ligand‐regulated interactions were validated in in vitro ELISA binding assays. AMPK binding LiRPs were then tested for modulation of AMPK kinase activity using purified AMPK in in vitro kinase assays. We found that the LiRPs inhibited the kinase activity of AMPK in a ligand regulated fashion, inhibiting the AMP stimulated state to a much greater extent. These results demonstrate the use of our ligand‐regulated peptide system and suggest the possibility of a novel regulatory peptide/protein‐binding site for AMPK.