Phosphoinositide-dependent kinase-1 (PDK1) mediates activation of many AGC kinases by docking onto a phosphorylated hydrophobic motif located C-terminal of the catalytic domain in the AGC kinase. The interaction shifts PDKI into a conformation with increased catalytic activity and leads to autophosphorylation of PDKI. We demonstrate here that addition of a hydrophobic motif peptide increases the catalytic activity of PDKI orthologues from Homo sapiens, Aplysia californica, Arahidopsis thaliana, Schizosaccharomyces pombe (ksgl), and Saccharomyces cerevisiae (Pkh 1 and Pkh2) 2- to 12-fold. Furthermore, the hydrophobic motif peptide increases autophosphorylation of PDKI from Homo sapiens, S. pombe, and S. cerevisiae (Phk2). Our results suggest that PDKI interaction and activation by the hydrophobic motif of AGC kinases is a central mechanism in PDKI function, which is conserved during eukaryotic evolution. (C) 2004 Elsevier Inc. All rights reserved.