Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase dom ain of PDK1 interacts with a region of protein kinase C-related kinase-2 (P RK2), termed the PDK1-interacting fragment (PIF), through a hydrophobic mot if, Here we identify a hydrophobic pocket in the small lobe of the PDK1 kin ase domain, separate from the ATP- and substrate-binding sites, that intera cts with PIF, Mutation of residues predicted to form part of this hydrophob ic pocket either abolished or significantly diminished the affinity of PDK1 for PIF, PIF increased the rate at which PDK1 phosphorylated a synthetic d odecapeptide (T308tide), corresponding to the sequences surrounding the PDK 1 phosphorylation site of PKB, This peptide is a poor substrate for PDK1, b ut a peptide comprising T308tide fused to the PDK1-binding motif of PIF was a vastly superior substrate for PDK1, Our results suggest that the PIF-bin ding pocket on the kinase domain of PDK1 acts as a 'docking site', enabling it to interact with and enhance the phosphorylation of its substrates.