Fine tuning PDK1 activity by phosphorylation at Ser163

3-Phosphoinositide-dependent protein kinase-1 ( PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser(244) in the activation loop and at a novel site: Ser(163) in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser(163) of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser(163) with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr(149) and Gln(223). In support of this, mutation of Tyr(149) to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser(163) may provide a mechanism to fine-tune PDK1 activity and function in cells.