pervanadate and Glioblastoma

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a approximately 10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.

Topics: 3T3-L1 Cells; Amino Acid Motifs; Animals; Cell Line; Cell Line, Tumor; Cell Membrane; Chromatography, Gel; DNA-Activated Protein Kinase; DNA-Binding Proteins; DNA, Complementary; Enzyme Activation; Genetic Complementation Test; Glioblastoma; Humans; Insulin; Mice; Microscopy, Fluorescence; Models, Biological; Nuclear Proteins; Phosphorylation; Plasmids; Precipitin Tests; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Serine; Time Factors; Transfection; Vanadates