Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells

Abstract

3-Phosphoinositide-dependent protein kinase-1 (PDK1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Cζ. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser399 and Thr516 as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser244 in the activation loop, are phosphorylated in cells and demonstrated that Ser244 is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser244, but not at Ser399 or Thr516, led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser399 or Thr516 is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1T516E, but not of mPDK-1S244E or mPDK-1S399D, in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr308 to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.