DNA-dependent protein kinase-mediated phosphorylation of protein kinase B requires a specific recognition sequence in the C-terminal hydrophobic motif.
DNA-dependent protein kinase (DNA-PK) has been implicated in a variety of nuclear processes including DNA double strand break repair, V(D)J recombination, and transcription. A recent study showed that DNA-PK is responsible for Ser-473 phosphorylation in the hydrophobic motif of protein kinase B (PKB/Akt) in genotoxic-stressed cells, suggesting a novel role for DNA-PK in cell signaling. Here, we report that DNA-PK activity toward PKB peptides is impaired in DNA-PK knock-out mouse embryonic fibroblast cells when compared with wild type. In addition, human glioblastoma cells expressing a mutant form of DNA-PK (M059J) displayed a lower DNA-PK activity when compared with glioblastoma cells expressing wild-type DNA-PK (M059K) when PKB peptide substrates were tested. DNA-PK preferentially phosphorylated PKB on Ser-473 when compared with its known in vitro substrate, p53. A consensus hydrophobic amino acid surrounding the Ser-473 phospho-acceptor site in PKB containing amino acids Phe at position +1 and +4 and Tyr at position -1 are critical for DNA-PK activity. Thus, these data define the specificity of DNA-PK action as a Ser-473 kinase for PKB in DNA repair signaling.
Park
Jongsun
J
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel CH-4058, Switzerland. insulin@cnu.ac.kr
Feng
Jianhua
J
Li
Yuwen
Y
Hammarsten
Ola
O
Brazil
Derek P
DP
Hemmings
Brian A
BA
D000818:Animals; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D027041:Somatic Hypermutation, Immunoglobulin / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010455:Peptides; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C497264:Prkdc protein, mouse; D051057:Proto-Oncogene Proteins c-akt