Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

3-Phosphoinositide-dependent protein kinase-1 (PDK1) expressed in unstimula ted 293 cells was phosphorylated at Ser-25, Ser-241, Ser-393, Ser-396 and S er-410 and the level of phosphorylation of each site was unaffected by stim ulation with insulin-like growth factor-1. Mutation of Ser-241 to Ala aboli shed PDK1 activity, whereas mutation of the other phosphorylation sites ind ividually to Ala did not affect PDK1 activity. Ser-241, unlike the other ph osphorylation sites on PDK1, was resistant to dephosphorylation by protein phosphatase 2A(1), Ser-241 lies in the activation loop of the PDK1 kinase d omain between subdomains VII and VIII in the equivalent position to the sit e that PDK1 phosphorylates on its protein kinase substrates. PDK1 expressed in bacteria was active and phosphorylated at Ser-241, suggesting that PDK1 can phosphorylate itself at this site, leading to its own activation.