CHARACTERIZATION OF A 3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN-KINASE WHICH PHOSPHORYLATES AND ACTIVATES PROTEIN-KINASE B-ALPHA

Background: Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth fa ctors, and much of the current interest in this enzyme stems from the observation that it lies 'downstream' of phosphoinositide 9-kinase on intracellular signalling pathways, We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of PKB at two residues, Thr308 and Ser473. The phosphorylation of both residues is r equired for maximal activation of PKB. The kinases that phosphorylate PKB are, however, unknown. Results: We have purified 500 000-fold from rabbit skeletal muscle extracts a protein kinase which phosphorylates PKB alpha at Thr308 and increases its activity over 30-fold, We teste d the kinase in the presence of several inositol phospholipids and fou nd that only low micromolar concentrations of the D enantiomers of eit her phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P-3) or Ptd lns(3,4)P-2 were effective in potently activating the kinase, which ha s been named Ptdlns(3,4,5)P-2-dependent protein kinase-1 (PDK1). None of the inositol phospholipids tested activated or inhibited PKB alpha or induced its phosphorylation under the conditions used. PDK1 activit y was not affected by wortmannin, indicating that it is not likely to be a member of the phosphoinositide 3-kinase family. Conclusions: PDK1 is likely to be one of the protein kinases that mediate the activatio n of PKB by insulin and growth factors, PDK1 may, therefore, play a ke y role in mediating many of the actions of the second messenger(s) Ptd lns(3,4,5)P-3 and/or Ptdlns(3,4)P-2. (C) Current Biology Ltd.