title: Effect of the inositol polyphosphate InsP(6) on DNA-PK-dependent phosphorylation.
abstract: Inositol hexakisphosphate (InsP(6)) is a member of the inositol polyphosphate group that participates in numerous intracellular signaling pathways. Cheung and colleagues previously reported that InsP(6) stimulated double-strand break repair by nonhomologous end joining (NHEJ) in cell-free extracts and that InsP(6) binding by the Ku70/80 subunit of the DNA-dependent protein kinase (DNA-PK) was required for stimulation of NHEJ in vitro. This report describes InsP(6)-dependent phosphorylation of two NHEJ factors, XRCC4 and XLF, in partially purified human cell extracts. XRCC4 and XLF are known substrates for DNA-PK, which does not require InsP(6) for protein kinase activity. Consistent with a role for DNA-PK in these reactions, InsP(6)-dependent phosphorylation of XRCC4 and XLF was DNA dependent and not observed in the presence of DNA-PK inhibitors. Depletion of the Ku70/80 DNA-, InsP(6)-binding subunit of DNA-PK resulted in loss of InsP(6)-dependent phosphorylation and showed a requirement for Ku70/80 in these reactions. Complementation of Ku70/80-depleted reactions with recombinant wild-type Ku70/80 restored InsP(6)-dependent phosphorylation of XRCC4 and XLF. In contrast, addition of a Ku70/80 mutant with reduced InsP(6) binding failed to restore InsP(6)-dependent phosphorylation. While additional protein kinases may participate in InsP(6)-dependent phosphorylation of XRCC4 and XLF, data presented here describe a clear requirement for DNA-PK in these phosphorylation events. Furthermore, these data suggest that binding of the inositol polyphosphate InsP(6) by Ku70/80 may modulate the substrate specificity of the phosphoinositide-3-kinase-related protein kinase DNA-PK.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, College of Pharmacy, 1601 Parkview Avenue, N-301, Rockford, IL 61107, USA. Hanakahi@uic.edu
authors.forename: Les
authors.initials: L
authors.lastname: Hanakahi
mesh_terms: D000818:Animals; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits
chemical_list: D004268:DNA-Binding Proteins; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases