Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase.
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.
Feng
Jianhua
J
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel CH-4058, Switzerland.
Park
Jongsun
J
Cron
Peter
P
Hess
Daniel
D
Hemmings
Brian A
BA
D041721:3T3-L1 Cells; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D005816:Genetic Complementation Test; D005909:Glioblastoma / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009687:Nuclear Proteins; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007328:Insulin; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C076715:pervanadate; D014638:Vanadates; D012694:Serine; C494918:AKT1 protein, human; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C497264:Prkdc protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt